Soluble, stable form of HDM2, crystalline forms thereof and methods of use thereof

ABSTRACT

The present invention discloses modified Hdm2 proteins that are soluble. In addition, the present invention discloses nucleic acids that encode the modified Hdm2 proteins of the present invention. The invention also provides crystals of modified Hdm2 proteins that are suitable for X-ray crystallization analysis. The present invention also discloses methods of using the modified Hdm2 proteins and crystals thereof to identify, select and/or design compounds that may be used as anticancer agents. The present invention further discloses compounds that bind to modified Hdm2 proteins in protein-ligand complexes.

This application claims the benefit of U.S. Provisional Application60/461,787, filed Apr. 10, 2003, and 60/547,265, filed Feb. 24, 2004.These applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to a soluble and stable form of humanDouble Minute 2 protein, Hdm2. The present invention further pertains tonucleic acids encoding these proteins. The present invention alsorelates to a process of obtaining specific samples of Hdm2 that areamenable to forming homogeneous crystals for X-ray crystallizationanalysis and the crystals formed thereby. The present invention alsopertains to methods of using the X-ray diffractable crystals instructure-based drug design to identify compounds that can modulate theactivity of the protein.

BACKGROUND OF THE INVENTION

The most commonly inactivated tumor suppressor gene in human cancerencodes the p53 protein, a transcription factor that is intimatelyinvolved in maintaining the integrity of the genome in a cell [Hall andPeters, Adv. Cancer Res., 68:67-108 (1996); Hainaut et al., Nucleic AcidRes., 25:151-157 (1997); Sherr, Cancer Res., 60:3689-95 (2000)]. Inresponse to oncogenic stress signals, the cell triggers the p53transcription factor to initiate either apoptosis or cell cycle arrest.Apoptosis facilitates the elimination of damaged cells from theorganism, while cell cycle arrest enables damaged cells to repairgenetic damage [reviewed in Ko et al., Genes & Devel. 10:1054-1072(1996); Levine, Cell 88:323-331 (1997)]. The loss of the safeguardfunctions of p53 predisposes damaged cells to progress to a cancerousstate. Inactivating p53 in mice consistently leads to an unusually highrate of tumors [Donehower et al., Nature, 356:215-221 (1992)].

The p53 transcription factor promotes the expression of a number of cellcycle regulatory genes, including the gene encoding the Mouse DoubleMinute (Mdm2) protein [see, Chene, Nature Reviews Cancer 3:102-109(2003)]. The Mdm2 protein (designated Hdm2 in humans and Mdm2 in mice)acts to down-regulate p53 activity in an auto-regulatory manner [Wu etal, Genes Dev., 7:1126-1132 (1993); Bairak et al., EMBO J, 12:461-468(1993)]. In the absence of oncogenic stress signals, i.e., under normalcellular conditions, the Mdm2 protein serves to maintain p53 activity atlow levels [Wu et al, Genes Dev., 7:1126-1132 (1993); Barak et al., EMBOJ, 12:461-468 (1993)].

Interestingly, whereas Mdm2 negative (Mdm2^(−/−)) mice are not viable[Jones et al, Nature, 378:206-208 (1995); Montes de Oca Luna et al.,Nature, 378:203-206 (1995)], additional inactivation of the p53 generescues Mdm2^(−/−) mice [Jones et al, Nature, 378:206-208 (1995); Montesde Oca Luna et al., Nature, 378:203-206 (1995)]. These results indicatethat the misregulation of the p53 transcription factor in the Mdm2negative mice is the root cause of the observed lethality of theMdm2^(−/−) genotype, and that the regulation of p53 function relies onan appropriate balance between the two components of this p53-Mdm2auto-regulatory system. Indeed, this balance appears to be essential forcell survival.

There are at least three ways that Mdm2 acts to downregulate p53activity. First, Mdm2 can bind to the N-terminal transcriptionalactivation domain of p53 to block expression of p53-responsive genes[Kussie et al., Science, 274:948-953 (1996); Oliner et al., Nature,362:857-860 (1993); Momand et al, Cell, 69:1237-1245 (1992)]. Second,Mdm2 shuttles p53 from the nucleus to the cytoplasm to facilitate theproteolytic degradation of p53 [Roth et al, EMBO J, 17:554-564 (1998);Freedman et al., Mol Cell Biol, 18:7288-7293 (1998); Tao and Levine,Proc. Natl. Acad. Sci. 96:3077-3080 (1999)]. Finally, Mdm2 possesses anintrinsic E3 ligase activity for conjugating ubiquitin to p53 within theubiquitin-dependent 26S proteosome pathway [Honda et al., FEBS Lett,420:25-27 (1997); Yasuda, Oncogene 19:1473-1476 (2000)]. Thus, Mdm2impedes the ability of the p53 transcription factor to promote theexpression of its target genes through binding p53 in the nucleus.

Attenuating the p53-Mdm2 auto-regulatory system can have a criticaleffect on cell homeostasis. Consistently, a correlation between theoverexpression of Mdm2 and tumor formation has been reported [Chene,Nature 3:102-109 (2003)]. Since Mdm2 acts as a post-translationalregulatory effector of the p53 transcription factor, compounds thathinder the ability of Hdm2/Mdm2, to interact with p53 would beanticipated to cause an immediate increase in p53 activity, and therebyrapidly promote either cell cycle arrest or apoptosis in damaged cells.Not surprisingly then, there is currently a substantial effort beingmade to identify new anticancer agents that hinder the ability of Hdm2to interact with p53 [Chene, Nature 3:102-109 (2003)]. However, to date,no suitable anticancer agent has been found.

Structure-based drug design is one way to optimize the success ofidentifying useful antagonists of Hdm2, but use of this powerfulmethodology requires the three-dimensional structure of the targetprotein. So far, little information has been provided regarding thethree-dimensional structure of Hdm2. Indeed, the only structures of Mdm2currently available are those of Hdm2 and Xenopus Mdm2 (XMdm2),complexed with a p53 peptide, but neither crystalline form is suitablefor structure-based drug design [Kussie, et al. Science, 274(5289):948-953 (1996)]. Moreover, most of the protein-protein contacts in thesecrystal lattices are formed through the interaction of the exposedresidues in the bound p53 peptide (D21, K24, and L25), making the p53peptide difficult to displace, which makes it inaccessible to potentialinhibitors.

In direct contrast, a successful structure-based drug design programfocusing on Hdm2 requires a form of the Hdm2 protein that is amenable tocrystallization in the absence of any particular binding partner.Further, the crystal form of the p53-binding pocket of the Hdm2 proteinshould be accessible to potential inhibitors used for testing binding orfor co-structural determination. However, up until now, the solubilityand stability of the free Hdm2 protein has been significantly less thanthat of the Hdm2-p53 peptide complex.

Thus, there is a need to obtain nucleic acids that encode an Hdm2protein that is soluble and stable at high protein concentrations evenwhen the protein is free of p53 or fragments thereof. In addition, thereis a need to design purification procedures that lead to the preparationof an isolated active Hdm2 protein that is soluble and stable whenindependent of p53 or fragments thereof. In addition, there is a need toobtain reproducible crystals of Hdm2 that are of sufficient quality forX-ray crystallization analyses and structural determinations. There isalso a need to provide methods for identifying inhibitors of Hdm2through structure-based drug design and for combining potentialinhibitors with the crystals of Hdm2 and analyzing their binding.Further, there is a need to obtain Hdm2 protein samples that, whencombined with potential inhibitors, are amenable to forming homogenouscrystals.

SUMMARY OF THE INVENTION

The present invention provides modified Hdm2 proteins that are amenableto crystallization and are soluble in E. coli extracts. The presentinvention further discloses a set of amino acid substitutions of theHdm2 protein that improve its solubility and/or stability withoutcompromising its ability to bind p53. It is a further object of thepresent invention to provide a modified Hdm2 protein having an aminoacid substitution at one or more of the seven sites defined in Table 1.In one embodiment, the modified Hdm2 protein comprises the amino acid ofSEQ ID NO: 4, wherein one or more of the seven specific amino acidresidues denoted as X₁-X₇ of SEQ ID NO: 4 differs from that of wild-typeHdm2(17-125) (SEQ ID NO: 2) or said amino acid sequence comprising oneor more conservative amino acid substitutions at sites other than thatof X₁-X₇ of SEQ ID NO: 4. In other embodiments, the modified Hdm2protein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 6, 8 10 and 12; or said amino acid sequencecomprising one or more conservative amino acid substitutions at sitesother than that of X₁-X₇ of SEQ ID NO: 4.

The present invention further provides isolated and/or recombinantnucleic acids that encode the modified Hdm2 proteins of the presentinvention, as well as specific peptide fragments and fusion proteinsthereof. In one embodiment, the nucleic acid encodes a modified Hdm2protein comprising the amino acid of SEQ ID NO: 4, wherein one or moreof the seven specific amino acid residues denoted as X₁-X₇ of SEQ ID NO:4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2) or saidamino acid sequence comprising one or more conservative amino acidsubstitutions at sites other than that of X₁-X₇ of SEQ ID NO: 4. Inother embodiments, the nucleic acid encodes a modified Hdm2 proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 6, 8 10 and 12; or said amino acid sequence comprising oneor more conservative amino acid substitutions at sites other than thatof X₁-X₇ of SEQ ID NO: 4. In certain embodiments, the nucleic acid maycomprise a nucleotide sequence selected from the group consisting of SEQID NOS: 5, 7, 9 and 11. The present invention further providesexpression vectors that can comprise any of the nucleic acids of thepresent invention and a transcriptional control sequence. Preferably thenucleic acids of the present invention are operatively linked to atranscriptional control sequence in expression vectors. Host cellscomprising the expression vectors are also part of the presentinvention. In one particular embodiment, the host cell is an E coli,cell.

In addition, the present invention provides methods for producing theabove-mentioned modified Hdm2 proteins. One such embodiment comprisesculturing a host cell of the present invention that expresses a nucleicacid encoding a modified Hdm2 protein of the present invention, therebyproducing the modified Hdm2 protein. Methods for purifying and/orobtaining the resulting recombinant modified Hdm2 proteins are alsoincluded in the present invention, as are the purified recombinantmodified Hdm2 proteins.

The present invention further provides compounds that bind to Hdm2. Inone such embodiment the compound is an acetylated tripeptide. In aparticular embodiment of this type the compound is Ac-^(6Cl)WAC_(3c)E.In another embodiment the compound is Ac-^(6Br)WAC_(3c)E.

The present invention further provides protein-ligand complexes betweenthe modified Hdm2 proteins of the present invention and their ligands.Preferred ligands in the complex are Ac-^(6Cl)WAC_(3c)E andAc-^(6Br)WAC_(3c)E.

Crystals comprising a modified Hdm2 protein, and/or one of theprotein-ligand complexes of the present invention, also are part of thepresent invention. Preferably, such crystals effectively diffract X-raysfor the determination of the atomic coordinates of the protein and/or ofthe protein-ligand complex to a resolution of greater than 5.0 Å (e.g.,at least 3.0 Å, at least 2.5 Å, at least 2.0 Å, or at least 1.5 Å).

The invention provides a crystal comprising a polypeptide selected from(a) a modified Hdm2 protein, characterized by structural coordinatescomprising a root mean square deviation of conserved residue backboneatoms of less than about 1.5 Å (e.g., less than about 1.0 Å, less thanabout 0.5 Å, or less than about 0.1 Å) when superimposed on backboneatoms described by structural coordinates of Table 3 or (b) a modifiedHdm2 protein characterized by structural coordinates comprising a rootmean square deviation of conserved residue backbone atoms of less thanabout 1.5 Å (e.g., less than about 1.0 Å, less than about 0.5 Å, or lessthan about 0.1 Å) when superimposed on backbone atoms described bystructural coordinates of Table 4. In certain embodiments, the crystalmay be complexed with a compound that binds to modified Hdm2 (e.g.,Ac-^(6Cl)WAC_(3c)E or Ac-^(6Br)WAC_(3c)E)

The invention also provides the three-dimensional structure of themodified Hdm2 protein. In one embodiment, the three-dimensionalstructure is characterized by structural coordinates comprising a rootmean square deviation of conserved residue backbone atoms of less thanabout 1.5 Å (e.g., less than about 1.0 Å, less than about 0.5 Å, or lessthan about 0.1 Å) when superimposed on backbone atoms described bystructural coordinates of Table 3 or 4. The present invention furtherprovides methods of using this three-dimensional structural informationin drug discovery and/or to solve corresponding structures of Hdm2homologues, other crystalline forms of Hdm2 mutants, and co-complexes ofHdm2 and its ligands.

In another aspect of the present invention, methods are provided forobtaining a crystal comprising a modified Hdm2 protein. In oneembodiment, the crystal is obtained by vapor diffusion.

In another aspect, the present invention provides a crystalline form ofHdm2 protein that is amenable to ligand soaking experiments, whichenables X-ray crystallographic structural determinations to be performedon multiple Hdm2-ligand complexes in rapid succession.

The present invention further provides methods of obtaining a crystalcomprising a protein-ligand complex between a ligand and a polypeptidecomprising a modified Hdm2 protein. The present invention furtherprovides methods for exchanging ligands within a crystal.

In yet another aspect, the present invention provides a method fordesigning, selecting and/or optimizing a compound and then evaluating itfor use as an inhibitor of Hdm2. One such embodiment comprises obtaininga set of atomic coordinates that define the three-dimensional structureof the modified Hdm2 protein from a crystal of the present invention. Ina related embodiment, a set of atomic coordinates that define thethree-dimensional structure of the protein-ligand binding complex from acrystal of the present invention is obtained. In either case, apotential agent is then designed, selected or optimized by performingstructure-based drug design with the atomic coordinates obtained.Preferably, the design or selection is performed in conjunction withcomputer modeling.

In another aspect, the invention provides a method for evaluating theability of a potential inhibitor to associate with Hdm2 comprisingemploying computational means to perform a fitting operation between thepotential inhibitor and the structure coordinates of Hdm2 andquantitating the association between the potential inhibitor and Hdm2.

A compound that is predicted to inhibit Hdm2 can be synthesized, ifnecessary, and subsequently contacted with Hdm2 or an active fragmentthereof. The activity of the compound is then determined by an assaythat measures one or more of Hdm2's activities, as described above. Acompound that is predicted to inhibit Hdm2 is identified as an inhibitorof Hdm2 when there is a decrease in the activity of Hdm2 in the presenceof the agent relative to in its absence.

In a related aspect of the present invention, a computer is providedthat comprises a three-dimensional representation of a modified Hdm2protein in computer memory. One such computer comprises amachine-readable data storage medium comprising a data storage materialencoded with machine-readable data, comprising the atomic coordinates ofTable 3 or 4. In another embodiment, the computer comprises amachine-readable data storage medium comprising a data storage materialencoded with machine-readable data, comprising the atomic coordinates ofTable 3 or 4. In yet another embodiment, the computer comprises amachine-readable data storage medium comprising a data storage materialencoded with machine-readable data, comprising the atomic coordinates ofTable 3 or 4. Preferably, the computer further comprises a workingmemory for storing instructions for processing the machine-readabledata, a central-processing unit coupled to the working memory and to themachine-readable data storage medium for processing the machine readabledata into a three-dimensional representation of the modified Hdm2protein and/or Hdm2 protein-ligand complex. More preferably, thecomputer includes a display coupled to the central-processing unit fordisplaying the three-dimensional representation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides stable modified Hdm2 proteins produced byintroducing an amino acid substitution into one or more of a unique setof amino acid residues of Hdm2. The modified Hdm2 proteins of thepresent invention have an improved solubility and form novel crystalsthat heretofore were unattainable with the wild-type Hdm2 protein. Thepresent invention further provides methods for generating and purifyingthese modified Hdm2 proteins. The modified Hdm2 proteins of the presentinvention may be used for the structural determination of Hdm2 by X-raycrystallography and/or NMR.

Crystals of the modified Hdm2 protein and their resulting structures canbe used to design high affinity inhibitors of Hdm2 that may be used inthe treatment of cancer. Such drugs may be particularly useful to treatsoft-tissue tumors, osteosarcomas and oesophageal carcinomas.

Structure-based drug design is the most efficient method for such drugdevelopment. In one common paradigm, a three dimensional structure isdetermined for a protein, e.g., the modified Hdm2, and/or acorresponding protein-ligand complex. Potential antagonists (e.g.,inhibitors and/or potential drugs) of the protein are then identifiedand/or designed with the aid of computer modeling [Bugg et al.,Scientific American, December: 92-98 (1993); West et al., TIPS, 16:67-74(1995); Dunbrack et al., Folding & Design, 2:27-42 (1997)]. The drugcandidates are then selected and tested. The most promising drugcandidates are identified and then combined with the protein in acrystalline protein-ligand complex. The three-dimensional structure ofthe protein-ligand complex is then determined, and new potentialantagonists of the protein are identified and/or designed with the aidof computer modeling. This process can then be continued in successiveiterations until a lead drug candidate is identified.

Heretofore, the ability to perform structure based drug design with Hdm2was severely hampered due to the lack of a crystalline form of the Hdm2that is conducive for such studies. The expression and purification of amodified Hdm2 protein that when placed in a protein-ligand complex canform a monodisperse preparation, as disclosed herein, is thereforecritical for the initiation of a structure based drug design program.

In addition, the present invention provides two specific ligands forHdm2, the acetylated tripeptides, Ac-^(6Cl)WAC_(3c)E andAc-^(6Br)WAC_(3c)E. Their precise chemical structures are providedbelow. These tripeptides can be used to bind the modified Hdm2 proteinsof the present invention to form a protein-ligand complex that is thencrystallized. Such X-ray diffractable crystals can be used for structurebased drug design to identify anti-tumor drugs.

Patches of hydrophobic amino acid residues on the surface of the Hdm2protein were initially determined to be a critical factor leading to therelative insolubility of the wild-type Hdm2 protein. Interrupting thesehydrophobic patches by replacing selected surface hydrophobic amino acidresidues with hydrophilic amino acid alternatives were found to increasethe solubility of the ensuing modified Hdm2 protein, as well as createnew crystal contact sites.

The present invention therefore provides several approaches foridentifying appropriate hydrophobic amino acid residues to be replacedby more hydrophilic alternatives. One such approach entails employingthe Clustalw program to align the amino acid sequences of Hdm2 and Hdm4analogs from a number of species to identify natural amino acidvariations. In one particular alignment, protocol Hdm2 or Hdm4 sequencesfrom the following species were used: Brachydanio rerio (Zebrafish),Canis familiaris (Dog), Equus caballus (Horse), Homo sapiens (Human),Mesocricetus auratus (Golden Hamster), Mus musculus (Mouse), Xenopuslaevis (African Clawed Frog) and Gallus gallus (Chicken). Seven Hdm2surface amino acid residues in the hydrophobic patches of Hdm2 wereidentified and the preferred alternative amino acids at those sitesnoted, see Table 1 below. In a related approach, the seven amino acidsubstitutions were selected to specifically increase Hdm2 solubility,see modified Hdm2 (HK₅) having the amino acid sequence of SEQ ID NO: 12.

In still another approach, the amino acid sequences of Hdm2 and XMdm2were compared to identify the positions of surface, solvent exposed,hydrophobic amino acid residues of the human protein that were occupiedby more hydrophilic residues in the corresponding Xenopus laevis aminoacid sequence. From this protein surface analysis, four potential aminoacid substitutions were chosen: Human: Xenopus laevis: F55Y, Y76H,Y104S, V109S.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1996) (herein “Ausubel et al., 1996”).

As used herein the following terms shall have the definitions set outbelow:

As used herein the term “polypeptide” is used interchangeably with theterm “protein” and is further meant to encompass peptides. Therefore, asused herein, a polypeptide is a polymer of two or more amino acidsjoined together by peptide linkages. Preferably, a polypeptide is apolymer comprising twenty or more amino acid residues joined together bypeptide linkages, whereas a peptide comprises five to twenty amino acidresidues joined together by peptide linkages.

As used herein a polypeptide “consisting essentially of” or that“consists essentially of” a specified amino acid sequence is apolypeptide that

(i) retains an important characteristic of the polypeptide comprisingthat amino acid sequence, e.g., the ability to bind p53 and/or act as aligase for conjugating ubiquitin to p53, and

(ii) further comprises the identical amino acid sequence, except itconsists of plus or minus 10% (or a lower percentage), and preferablyplus or minus 5% (or a lower percentage) of the amino acid residues. Ina particular embodiment, additional amino acid residues included as partof the polypeptide are part of a linked Tag, such as a C-terminal His₆Tag.

The term “generic Mdm2” is used herein to refer to the double minute 2polypeptide from any species. When used by itself, Mdm2 refers to mouseMdm2. When used with another species name appended to it, it refers tothe Mdm2 ortholog from the named species, e.g., Xenopus Mdm2 refers tothe Mdm2 ortholog from Xenopus. “Mdm2” also encompass modified formsthereof.

As used herein the terms “human Mdm2” and “Hdm2” are usedinterchangeably to denote the human ortholog of Mdm2. “Hdm2” alsoencompass modified forms thereof. Hdm2 has the GenBank accession numberof M92424. Mouse Mdm2 has the GenBank accession number of X58876 [seealso, published U.S. patent application 2002/0045192, published Apr. 18,2002, the contents of which are hereby incorporated by reference intheir entirety.] The amino acid sequences provided herein correspond toamino acid residues 17-125 of the full-length Hdm2 protein and retaintheir numerical designation from that full-length sequence. Therefore,the first amino acid residue of the wild-type Hdm2(17-125) sequence, SEQID NO: 2, corresponds to amino acid 17 of the full-length Hdm2 protein.

As used herein a “modified Hdm2” is identical to the wild-typeHdm2(17-125) except it has at least one amino acid substitution, i.e.,it has one or more amino acid substitutions. Furthermore, a modifiedHmd2 of the present invention comprises an amino acid substitution atone or more of the seven positions listed in Table 1 and denoted in SEQID NO: 4. Preferably, that amino acid substitution is one that isspecifically defined in Table 1 (and denoted in SEQ ID NO: 4). It isalso preferable that a modified Hdm2 protein of the present inventioncomprises 109 amino acid residues and that the positions of these 109residues correspond to amino acid residues 17-125 of the full-lengthwild-type Hdm2 protein.

As used herein, a “conservative amino acid substitution” is thesubstitution of a functionally equivalent amino acid for an amino acidwithin the sequence of Hdm2. The conservative amino acid substitutioncan be at any position in the Hdm2 sequence except for the sevenpositions identified in Table 1, for which the alternatives arespecifically defined. In general, a functionally equivalent amino acidis one having a similar polarity and/or molecular properties for theamino acid within the sequence. Specifically, an amino acid substitutemay be selected from other members of the class to which the amino acidbelongs. The class of nonpolar amino acids includes alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The class of polar neutral amino acids includes glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The class ofpositively charged (basic) amino acids includes arginine, and lysine.The negatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Particularly preferred conserved amino acid exchanges are:

(a) Lys for Arg or vice versa such that a positive charge may bemaintained;

(b) Glu for Asp or vice versa such that a negative charge may bemaintained;

(c) Ser for Thr or vice versa such that a free —OH can be maintained;

(d) Gln for Asn or vice versa such that a free NH₂ can be maintained;and

(e) Ile for Leu or for Val or vice versa as roughly equivalenthydrophobic amino acids.

As used herein the term “specific peptide fragment” is a peptide thatcomprises at least six amino acid residues, and preferably at leasttwelve amino acid residues of a modified Hdm2 protein that differs fromthe corresponding fragment of the wild-type Hdm2 by at least one aminoacid residue. Furthermore, the different amino acid residue (ordifferent residues) is located at a position that corresponds to one (ormore) of the seven variable sites denoted in SEQ ID NO: 4 and listedTable 1 below.

As used herein the term “chimeric” protein is meant to include fusionproteins. “Chimeric” proteins of the present invention comprise at leasta portion of a non-Hdm2 protein or peptide joined via a peptide bond toat least a portion of an Hdm2 protein, preferably a modified Hdm2.Chimeric proteins can have additional structural, regulatory, and/orcatalytic properties. As used herein a chimeric protein can containmultiple additions to at least a portion of a modified Hdm2 protein,e.g., it can comprise both a His₆Tag and a signal sequence. In aparticular embodiment the chimeric protein functions as a means ofdetecting and/or isolating the polypeptide or fragment thereof after arecombinant nucleic acid encoding the modified Hdm2 protein or fragmentthereof is expressed. Non-Hdm2 amino acid sequences are preferablyeither amino- or carboxy-terminal to the modified Hdm2 sequence.

As used herein, DNA and protein sequence percent identity can bedetermined using C, MacVector 6.0.1, Vector NTI (Informax, Inc. MD),Oxford Molecular Group PLC (1996) and the Clustal W algorithm with thealignment default parameters, and default parameters for identity. Thesecommercially available programs can also be used to determine sequencesimilarity using the same or analogous default parameters.Alternatively, an Advanced Blast search under the default filterconditions can be used, e.g., using the GCG (Genetics Computer Group,Program Manual for the GCG Package, Version 7, Madison, Wis.) pileupprogram using the default parameters.

As used herein a “nucleic acid” refers to the phosphate ester polymericform of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. When referring to anucleic acid that is double stranded both the “sense” strand and thecomplementary “antisense” strand are intended to be included. Thus anucleic acid that is hybridizable to SEQ ID NO: 1, for example, can beeither hybridizable to the “sense” strand of SEQ ID NO: 1, which isparticularly listed in the SEQUENCE LISTING, or to the “antisense”strand which can be readily determined from that SEQUENCE LISTING.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which can then be trans-RNAspliced and translated into the protein encoded by the coding sequence.

A nucleic acid sequence is “operatively linked” to an expression controlsequence when the expression control sequence controls or regulates thetranscription and translation of that nucleic acid sequence. The termoperatively linked includes having an appropriate start signal.

A “heterologous nucleotide sequence” as used herein is a nucleotidesequence that is added by recombinant methods to a nucleotide sequenceencoding a modified Hdm2 of the present invention or encoding a fragmentthereof, to form a nucleic acid that is not naturally formed in nature.Such nucleic acids can encode chimeric proteins. In addition, as usedherein, a heterologous nucleotide sequence need not be a singlecontiguous nucleotide sequence, but can include multiple non-contiguousnucleotide sequences that have been combined with a nucleotide sequenceencoding a modified Hdm2 protein of the present invention, or a portionthereof. A heterologous nucleotide sequence can comprise non-codingsequences including restriction sites, regulatory sites, promoters andthe like. In still another embodiment the heterologous nucleotide canfunction as a means of detecting a nucleotide sequence of the presentinvention. The present invention provides heterologous nucleotidesequences that, when combined with nucleotide sequences encoding amodified Hdm2 protein or a fragment thereof, are necessary andsufficient to encode all of the chimeric proteins of the presentinvention.

The phrase “binding to” in regard to a ligand binding to a polypeptideis used herein to include any or all such specific interactions thatlead to a protein-ligand binding complex. This can include processessuch as covalent, ionic (electrostatic and/or charged), hydrophobic andhydrogen bonding, but does not include non-specific associations suchsolvent preferences.

As used herein a “ligand” of a Mdm2 protein, e.g., a modified Hdm2protein, is a compound that binds to the polypeptide in a protein-ligandbinding complex. In a specific embodiment of the present invention theligand inhibits the ability of the Mdm2 protein to bind p53 when theligand is bound to the Mdm2 protein in a protein-ligand binding complex.In another embodiment, the ligand inhibits the ability of Mdm2 to act asan E3 ligase for conjugating ubiquitin to p53 when the ligand is boundto the Mdm2 protein in a protein-ligand binding complex. Such ligandsmay also be termed an “inhibitor”.

As used herein, a “protein-ligand binding complex” orpolypeptide-compound complex” is a specific association between apolypeptide and the compound that binds to it. In a preferred embodimentof the present invention, the ligand or compound is an inhibitor of thepolypeptide. In a particular embodiment of this type, the binding of theinhibitor to the polypeptide occurs at the active site of thepolypeptide.

As used herein “incubating a ligand with a crystal” is usedinterchangeably with “soaking a crystal with a ligand”. Incubating aligand with a crystal is the contacting of a ligand with a crystal of apolypeptide under the appropriate conditions and for a sufficient timeperiod (e.g., hours to several days) for the ligand to bind to thecrystalline polypeptide and form a crystalline protein-ligand complex.Such incubating can further include contacting an excess of a substituteligand with a crystal of a protein-ligand complex under the appropriateconditions and for a sufficient time period (e.g., hours to severaldays) for the substitute ligand to replace the initial ligand and formthe new crystalline protein-ligand complex.

As used herein the terms “displacing”, “replacing”, and “exchanging” areused interchangeably in regard to the substitution of one ligand in aprotein-ligand complex for another.

As used herein an “excess of a substitute ligand” is an amount of thatligand that is sufficient to replace 80% or more, and preferably 90% ormore, of the initial ligand in a protein-ligand complex. In a particularembodiment of this type, the concentration of the substitute ligand isabout ten-fold higher than the concentration of the protein-ligandcomplex. In a preferred embodiment, the concentration of the substituteligand is about one hundred-fold higher than the concentration of theprotein-ligand complex.

As used herein the term “X-ray diffractable crystal” is a crystal of acompound, e.g., a protein that yields a discernable diffraction patternwhen subjected to 0.5 to 2.5 Å incident X-ray radiation.

As used herein an “X-ray quality crystal” is an X-ray diffractablecrystal that can yield meaningful structural data of its crystallinecomposition when subjected to X-ray crystallographic analysis.

As used herein, and unless otherwise specified, the terms “agent”,“potential drug”, “compound”, or “test compound” are usedinterchangeably, and refer to chemicals that have or potentially have ause as a modulator of the activity of Mdm2. Preferably the modulator isan inhibitor of the binding complex formed between p53 and Mdm2.Preferably such agents include drugs for the treatment or prevention ofa disease and/or condition involving the p53 transcription factor, e.g.,cancer. Therefore, such agents may be used, as described herein, in drugassays and drug screens and the like.

As used herein a “small organic molecule” is an organic compound [ororganic compound complexed with an inorganic compound (e.g., metal)]that has a molecular weight of less than 3 Kd.

As used herein the terms “approximately” and “about” are used to signifythat a value is within twenty percent of the indicated value i.e., anamino acid sequence containing “approximately” 110 amino acid residuescan contain between 88 and 132 amino acid residues.

As used herein the phrases “structure based rational drug design”,“structure based drug design” and “structure assisted drug design” areused interchangeably. These phrases are meant to convey a particularmethod of identifying and/or designing a ligand (preferably aninhibitor) for a specific target protein that includes the use of thethree-dimensional structure of that protein and/or its correspondingprotein-ligand complex.

Nucleic Acids Encoding Mdm2 Proteins

The nucleic acids can further comprise heterologous nucleotidesequences. In one embodiment, the nucleic acid encodes a modified Hdm2protein comprising the amino acid of SEQ ID NO: 4, wherein one or moreof the seven specific amino acid residues denoted as X₁-X₇ of SEQ ID NO:4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2). In aparticular embodiment of this type the nucleic acid comprises thenucleotide sequence of SEQ ID NO: 3, wherein at least one nucleotide ofone of the codons encoding one or more of the seven specific amino acidresidues denoted as X₁-X₇ of SEQ ID NO: 4 differs from the nucleic acidsequence of wild-type Hdm2(17-125) (SEQ ID NO: 1) and wherein said codonencodes a different amino acid from that of wild-type Hdm2(17-125) (SEQID NO: 2).

In one embodiment, the nucleic acid encodes a modified Hdm2 proteincomprising the amino acid sequence of SEQ ID NO: 6. In a particularembodiment of this type, the nucleic acid comprises the nucleotidesequence of SEQ ID NO: 5. In another embodiment, the nucleic acidencodes a modified Hdm2 protein comprising the amino acid sequence ofSEQ ID NO: 8. In a particular embodiment of this type, the nucleic acidcomprises the nucleotide sequence of SEQ ID NO: 7. In still anotherembodiment, the nucleic acid encodes a modified Hdm2 protein comprisingthe amino acid sequence of SEQ ID NO: 10. In a particular embodiment ofthis type, the nucleic acid comprises the nucleotide sequence of SEQ IDNO: 9. In yet another embodiment, the nucleic acid encodes a modifiedHdm2 protein comprising the amino acid sequence of SEQ ID NO: 12. In aparticular embodiment of this type, the nucleic acid comprises thenucleotide sequence of SEQ ID NO: 11. Nucleic acids that consist of thenucleotide sequences that encode the modified Hdm2 proteins of thepresent invention or that consist of nucleotide sequences that encodespecific peptide fragments of those proteins are also provided. Thepresent invention also includes those nucleic acids that encode amodified Hdm2 comprising one or more conservative amino acidsubstitutions. In certain embodiments, there may be 1-11 conservativeamino acid substitutions, preferably 1, 2 or 3 conservative amino acidsubstitutions. In a related embodiment, the present invention providesnucleic acids that further comprise a heterologous nucleotide sequence.

Obtaining and/or constructing a cDNA that encodes a Mdm2 protein,including Hdm2 proteins and the modified Hdm2 proteins of the presentinvention, facilitates the production of the large quantities of proteinrequired to perform standard enzyme assays and/or X-ray crystallographicanalysis.

The present invention provides specific nucleic acid constructs thatallow for the expression and isolation of large quantities of stable andactive modified Hmd2 proteins. These nucleic acids can further containheterologous nucleotide sequences. To express a recombinant protein ofthe present invention in a host cell, an expression vector can beconstructed comprising the corresponding cDNA. The present inventiontherefore, provides expression vectors containing nucleic acids encodingthe modified Hmd2 proteins of the present invention. Due to thedegeneracy of nucleotide coding sequences, other DNA sequences thatencode the same or substantially the same amino acid sequence as anucleic acid encoding a modified Hmd2 protein of the present inventionmay be used in the practice of the present invention. These include, butare not limited to, allelic genes, homologous genes from other species,which are altered by the substitution of different codons that encodethe same amino acid residue within the sequence, thus producing a silentchange. Host cells comprising the expression vectors of the presentinvention are also provided. One particular host cell, an E. coli cell,is specifically exemplified below.

General methods for the cloning of cDNAs and expression of theircorresponding recombinant proteins have been described [see Sambrook andRussell, Molecular Cloning, A Laboratory Manual, 3 edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor L.I. (2000)]. The particularmethodology used herein is described in Example 1 below. Preferably, allof the nucleic acid constructs of the present invention are sequenceconfirmed.

Any technique for mutagenesis known in the art can be used to convertthe native (wild-type) Hdm2 to a modified Hdm2 of the present invention,including but not limited to, in vitro site-directed mutagenesis[Hutchinson et al., J. Biol. Chem., 253:6551 (1978); Zoller and Smith,DNA, 3:479-488 (1984); Oliphant et al., Gene, 44:177 (1986); Hutchinsonet al., Proc. Natl. Acad. Sci. U.S.A., 83:710 (1986)]. The use of TAB@linkers (Pharmacia), etc. and PCR techniques also can be employed forsite directed mutagenesis [see Higuchi, “Using PCR to Engineer DNA”, inPCR Technology: Principles and Applications for DNA Amplification, H.Erlich, ed., Stockton Press, Chapter 6, pp. 61-70 (1989)].

Preferably mutagenesis (i.e., modification) of an Hdm2 transcript isperformed in a two step process [Wang and Malcolm, BioTechniques26:680-682 (1999)]. In Examples 2 and 3 below, two extension reactionswere performed in separate tubes in the first stage: (i) one containingthe forward primer, and (ii) the other containing the reverse primer.After two cycles, the two reactions are mixed and the standardQuickChange mutagenesis procedure was carried out for an additional 18cycles. Following amplification, the parental strand was digested with 1Unit of Dpn1 for 2 hours and an aliquot was transformed into DH5-alphacells [GeneWiz, New York, N.Y.]. The pET32 Xa/LIC-hdm2 (17-125) vectorwas used as a template.

When the modified Hdm2 has three amino acid substitutions, as describedin the Example 1 below, the GENETAILOR Site-directed Mutagenesis system(Invitrogen, Carlsbad, Calif., USA) was used with the pET32 Xa/LIC-hdm2(17-125) as the template. In this case the GENETAILOR mutagenesis wasperformed according to the manufacture's instruction manual, with thelone exception being the use of the pfu turbo DNA polymerase.

The Modified Hdm2 Proteins

In one embodiment, the modified Hdm2 polypeptide of the inventioncomprises the amino acid sequence SEQ ID NO: 4, wherein one or more ofthe seven specific amino acid residues denoted as X₁-X₇ of SEQ ID NO: 4differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2). In anotherembodiment, the modified Hdm2 polypeptide comprises one or moreconservative amino acid substitutions at sites other than that of X₁-X₇of SEQ ID NO: 4. In certain embodiments, there may be 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 conservative amino acid substitutions in the sequence atsites other than those indicated in Table 1. In preferred embodiments,there may be 1, 2 or 3 conservative amino acid substitutions at sitesother than those indicated in Table 1.

In particular embodiments, the amino acid residues at all seven of thevariable positions in the amino acid sequence of SEQ ID NO: 4 aredifferent than that of SEQ ID NO: 2. In a specific embodiment of thistype, the modified Hdm2 protein is the Hdm2(HK5) comprising the aminoacid sequence of SEQ ID NO: 12 or said modified Hdm2 protein comprisingthe amino acid sequence of SEQ ID NO: 12 comprising one or moreconservative amino acid substitutions at sites other than that of X₁-X₇of SEQ ID NO: 4 as described above. In other embodiments, the modifiedHdm2 protein comprises the amino acid sequence SEQ ID NO: 4 wherein theamino acid residues at six, five, four or three of the seven variablepositions (X₁-X₇) in the amino acid sequence of SEQ ID NO: 4 aredifferent than that of SEQ ID NO: 2. In certain embodiments, themodified Hdm2 protein comprises one or more conservative amino acidsubstitutions at sites other than that of X₁-X₇ of SEQ ID NO: 4.

In yet another embodiment, the amino acid residues at two of the sevenvariable positions in the amino acid sequence of SEQ ID NO: 4 aredifferent than that of SEQ ID NO: 2. In a particular embodiment of thistype the modified Hdm2 protein is Hdm2(F55Y/Y76H) protein comprising theamino acid sequence of SEQ ID NO: 10 or said modified Hdm2 proteincomprising one or more conservative amino acid substitutions at sitesother than that of X₁-X₇ of SEQ ID NO: 4. In another embodiment, onlyone of the amino acid residues at the seven variable positions in theamino acid sequence of SEQ ID NO: 4 is different than that of SEQ ID NO:2. In a particular embodiment of this type, the modified Hdm2 protein isthe Hdm2(F55Y) protein comprising the amino acid sequence of SEQ ID NO:8 or said modified Hdm2 protein comprising one or more conservativeamino acid substitutions at sites other than that of X₁-X₇ of SEQ ID NO:4. In another particular embodiment, the modified Hdm2 protein isHdm2(Y76H) protein comprising the amino acid sequence of SEQ ID NO: 6 orsaid modified Hdm2 protein comprising one or more conservative aminoacid substitutions at sites other than that of X₁-X₇ of SEQ ID NO: 4.

The present invention further provides a modified Hdm2 proteinconsisting of, or consisting essentially of, SEQ ID NOs: 4, wherein oneor more of the seven specific amino acid residues denoted as X₁-X₇ ofSEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2).In certain embodiments, said modified Hdm2 protein comprises one or moreconservative amino acid substitutions at sites other than that of X₁-X₇of SEQ ID NO: 4. A modified Hdm2 protein is further provided thatconsists of, or consists essentially of, SEQ ID NOs: 6, 8, 10 or 12, orsaid modified Hdm2 protein comprising one or more conservative aminoacid substitutions at sites other than that of X₁-X₇ of SEQ ID NO: 4.

Fusion proteins that comprise the modified Hdm2 proteins of the presentinvention are also provided, as well as specific peptide fragments ofthose proteins.

In one such embodiment, the modified Hdm2 protein comprises the aminoacid sequence of SEQ ID NO: 4. In a preferred embodiment, the modifiedHdm2 protein comprises the amino acid sequence of SEQ ID NO: 6. In oneembodiment the compound is Ac-^(6Br)WAC_(3c)E. In a preferredembodiment, the compound is AC-^(6Cl)WAC_(3c)E.

The amino acid sequences of the wild-type (WT) Hdm2 and the followingmodified Hdm2 proteins corresponding to amino acid residues 17-125 ofthe full-length wild-type Hdm2 are provided below:

WT Hdm2(17-125): SEQ ID NO: 2     SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGT SVSEN ModifiedHdm2 (X₁-X₇): SEQ ID NO: 4      SQIPASEQETX ₁VRPKPX₂LLKLLKSVGAQKDTYTMKEVLX ₃YLGQYIMTK RLYDEKQQHIVX ₄CSNDX ₅LGDLFGVX₆SFSVKEHRKIYTMIX ₇RNLVVVNQQES SDSGTSVSEN Modified Hdm2 (Y76H): SEQ IDNO: 6      SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVHCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSG TSVSEN ModifiedHdm2 (F55Y): SEQ ID NO: 8     SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLYYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGT SVSEN ModifiedHdm2 (F55Y/Y76H): SEQ ID NO: 10     SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLYYLGQYIMTKRLYDEKQQHIVHCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSG TSVSEN ModifiedHdm2 (HK₅): SEQ ID NO: 12     SQIPASEQETKVRPKPKLLKLLKSVGAQKDTYTMKEVLHYLGQYIMTKRLYDEKQQHIVKCSNDKLGDLFGVKSFSVKEHRKIYTMIYRNLVVVNQQESSDS GTSVSEN

Modified Hdm2 (HK₅) as indicated above has six amino acid substitutions(L27K, L33K, F55H, Y76K, L81 K, and P89K) selected to specificallyincrease Hdm2 solubility; only two are derived from natural variants.

Column 1 of Table 1 denotes the seven variable amino acid positions inthe sequence of SEQ ID NO: 4, and presents the numbering of the aminoacid positions in relation to both the full-length wild-type Hdm2 andthe corresponding numbering of the wild-type Hdm2(17-125) having theamino acid sequence of SEQ ID NO: 2 (in parentheses). The seven definedpositions in SEQ ID NO: 4 in which the amino acid residue canspecifically vary are denoted as X₁-X₇ (column 2). The amino acidresidues in the full-length wild-type Hmd2 occupying those sevenpositions are listed in column 3. All of the natural variants identifiedby the Clustalw alignment of the amino acid sequences of Hdm2 and Hdm4analogs using Brachydanio rerio (Zebrafish), Canis familiaris (Dog),Equus caballus (Horse), Homo sapiens (Human), Mesocricetus auratus(Golden Hamster), Mus musculus (Mouse), Xenopus laevis (African ClawedFrog) and Gallus gallus (Chicken) are provided in column 4. Allacceptable amino acids are listed in Column 5, including the amino acidresidues for the seven respective positions of the wild-type Hmd2, whichare in bold.

TABLE 1 Amino Acid Substitutions for SEQ ID NO: 4 A.A. A.A. SpeciesPosition Name Hdm2 Variants Acceptable Variants  27 (11) X₁ L27 Q L, K,R, Q, E, D, S  33 (17) X₂ L33 Q L, K, R, Q, E, D, S  55 (39) X₃ F55 L,Y, H F, H, Y, K, R, Q, E, D, S  76 (60) X₄ Y76 H Y, H, K, R, Q, E, D, S 81 (65) X₅ L81 A, P, C L, K, R, Q, E, D, S, P, A  89 (73) X₆ P89 K, V,Q, T P, K, R, Q, E, D, S 104 (88) X₇ Y104 I, N, R, S Y, K, R, Q, E, D,S, N

In addition, the modified Hdm2 proteins of the present invention mayinclude conservative amino acid substitutions relative to the wild typesequence of Hdm2 (other than those at the seven positions identified inTable 1, for which the alternatives are specifically defined). Ingeneral, there are no more than 11 conservative amino acid substitutions(e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 conservativeamino acid substitutions). In a preferred embodiment, there are no morethan 3 conservative amino acid substitutions (0, 1, 2 or 3).

All of the modified Hdm2 proteins of the present invention also can bepart of a chimeric protein. In a specific embodiment, a chimericmodified Hdm2 protein is expressed in a prokaryotic cell. Such achimeric protein can be a fusion protein used to isolate a modified Hdm2protein of the present invention, through the use of an affinity columnthat is specific for the protein fused to the modified Hdm2 protein.Examples of such fusion proteins include: a glutathione-S-transferase(GST) fusion protein, a maltose-binding protein (MBP) fusion protein, aFLAG-tagged fusion protein, or a poly-histidine-tagged fusion protein.Specific linker sequences such as a Ser-Gly linker can also be part ofsuch a fusion protein. A chimeric modified Hdm2 protein of the presentinvention also can be expressed in a eukaryotic cell.

Expression of a chimeric, modified Hdm2 protein, or fragment thereof, asa fusion protein can facilitate stable expression, and/or allow forpurification based on the properties of the fusion partner. Thus thepurification of the recombinant polypeptides of the present inventioncan be simplified through the use of fusion proteins having affinityTags. For example, GST binds glutathione conjugated to a solid supportmatrix, MBP binds to a maltose matrix, and poly-histidine chelates to aNi-chelation support matrix [see Hochuli et al., Biotechnology6:1321-1325 (1998)]. The fusion protein can be eluted from the specificmatrix with appropriate buffers, or by treating with a protease that isspecific for a cleavage site that has been genetically engineered inbetween the modified Hdm2 protein and its fusion partner. Alternatively,a modified Hdm2 protein can be combined with a marker protein such asgreen fluorescent protein [Waldo et al., Nature Biotech. 17:691-695(1999); U.S. Pat. No. 5,625,048 and WO 97/26333].

Alternatively or in addition, other column chromatography steps (e.g.,gel filtration, ion exchange, affinity chromatography etc.) can be usedto purify the recombinant modified Hdm2 proteins of the presentinvention. In many cases, such column chromatography steps employ highperformance liquid chromatography or analogous methods in place of themore classical gravity-based procedures. The specific details for thepreferred purification procedure of the modified Hdm2 proteins of thepresent invention are provided in Example 1 below.

In addition, the modified Hdm2 proteins of the present invention andproteins thereof including, specific peptide fragment thereof can bechemically synthesized [see e.g., Synthetic Peptides: A User's Guide,W.H.Freeman & Co., New York, N.Y., pp. 382, Grant, ed. (1992)].

Crystallization of Protein

The modified Hdm2 proteins result in novel crystal forms not obtainedwith the wild-type protein. In one embodiment, the modified Hdm2 proteinmay be crystallized in the presence of one of a variety of differentcompounds (e.g., an small molecule inhibitor or a peptide from p53).Further, a compound complexed to modified Hdm2 may be exchanged in thecrystal by a second compound (e.g., a potential inhibitor) by soakingthe crystal in a solution containing the second compound. These crystalsand the resulting structures can be used to obtain a detailed view ofvarious inhibitors that bind to Hdm2, providing a basis for furtherdesign of potent inhibitors of Hdm2 to be used as anticancer agents.

Crystallization may be accomplished by using any of the known methods inthe art (Giegé, et al., (1994) Acta Crystallogr. D50: 339-350;McPherson, (1990) Eur. J. Biochem. 189: 1-23). Such techniques includemicrobatch, hanging drop, seeding and dialysis. Preferably, hanging-dropvapor diffusion (McPherson, (1976) J. Biol. Chem. 251: 6300-6303) ormicrobatch methods (Chayen (1997) Structure 5: 1269-1274) are used. Ineach of these methods, it is important to promote continued crystalgrowth after nucleation by maintaining a supersaturated solution. In themicrobatch method, polypeptide is mixed with precipitants to achievesupersaturation, the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane that is placed into a solution containing precipitant.Equilibration across the membrane increases the precipitantconcentration thereby causing the polypeptide to reach supersaturationlevels. It is desirable to use a modified Hdm2 protein preparationhaving a concentration of at least about 1 mg/mL and preferably about 5mg/mL to about 60 mg/mL, more preferably about 20 mg/mL to about 50mg/mL, even more preferably about 30 mg/mL to about 40 mg/mL.Crystallization may be achieved in precipitant solutions containingpolyethylene glycol 1000-20,000 (PEG; average molecular weight rangingfrom about 1000 to about 20,000 Da), preferably about 2000 to about 6000Da, more preferably about 5000 Da, with concentrations ranging fromabout 10% to about 50% (w/v). It may also be desirable to include aprotein stabilizing agent. If glycerol is chosen as the proteinstabilizing agent, it is preferably provided at a concentration rangingfrom about 0.5% to about 20%. A suitable salt, such as magnesiumchloride, potassium chloride, sodium chloride, lithium chloride orsodium citrate may also be desirable in the precipitant solution,preferably in a concentration ranging from about 1 mM to about 2000 mM.The precipitant is preferably buffered to a pH of from about 6.5 toabout 9.5, preferably about 7.5-8.5. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, Hepes, Tris, MES and acetate. Crystals routinely grow ata wide range of temperatures. It is, however, preferred that crystalsform at temperatures between about 2° C. and about 26° C.

The crystals of the present invention have a wide range of uses. Forexample, high quality crystals are suitable for X-ray or neutrondiffraction analysis to determine the three dimensional structure ofHdm2 and in particular to assist in the identification of the protein'seffector sites. Knowledge of these sites and solvent accessible residuesallow structure-based design and construction of agonists andantagonists for Hdm2.

In addition, crystallization itself can be used as a purificationmethod. In some instances, a polypeptide or protein crystallizes from aheterogeneous mixture into crystals. Isolation of such crystals byfiltration and/or centrifugation, followed by redissolving thepolypeptide affords a purified solution suitable for use in growinghigh-quality crystals that are preferred for diffraction analysis.

Once a crystal of the present invention is grown, X-ray diffraction datacan be collected. One method for determining structure with X-raydiffraction data includes use of synchrotron radiation, under standardcryogenic condition; however, alternative methods may also be used. Forexample, crystals can be characterized by using X-rays produced by aconventional source, such as a sealed tube or a rotating anode. Methodsof characterization include, but are not limited to, precessionphotography, oscillation photography and diffractometer data collection.

The crystallizable compositions provided by this invention may beamenable to X-ray crystallography for providing the three-dimensionalstructure of a Hdm2 polypeptide. The present invention includes crystalswhich effectively diffract X-rays for the determination of the atomiccoordinates of Hdm2 to a resolution of greater than about 5.0 Ångströms(e.g., about 4.5 Å, about 4.0 Å, about 3 Å, about 2.5 Å, about 2 Å,about 1 Å, about 0.5 Å, about 0.1 Å), preferably greater than about 4.0Ångströms (e.g., about 3 Å, about 2.5 Å, about 2 Å, about 1 Å, about 0.5Å, about 0.1 Å), more preferably greater than about 2.8 Ångströms (e.g.,about 2.5 Å, about 2 Å, about 1 Å, about 0.5 Å, about 0.1 Å) and mostpreferably greater than about 2.0 Ångströms (e.g., about 1.5 Å, about1.0 Å, about 0.5 Å, about 0.1 Å).

The present invention includes Hdm2 crystals whose three-dimensionalstructure is described by the structure coordinates set forth in Table 3or 4. The scope of the present invention also includes crystals whichpossess structural coordinates which are similar to those set forth inTable 3 or 4; preferably, the crystals or the soluble polypeptides whichare used to form the crystals exhibit Hdm2 catalytic activity and/or p53binding (see above). In some embodiments, the crystal comprises apolypeptide that comprises the amino acid sequence of SEQ ID NO: 4,wherein one or more of the seven specific amino acid residues denoted asX₁-X₇ of SEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQID NO: 2). In other embodiments, the crystal comprises a polypeptidethat comprises the amino acid sequence selected from the groupconsisting of SEQ ID NOS: 8 and 12. In another embodiment, the modifiedHdm2 polypeptide comprises one or more conservative amino acidsubstitutions at sites other than that of X₁-X₇ of SEQ ID NO: 4.Structural similarity between crystals is discussed in detail below.

The term “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a beam of X-rays by the atoms (scattering centers) of amolecule. The diffraction data are used to calculate electron densitymaps and to establish the positions of the individual atoms of themolecule.

Those of skill in the art will understand that a set of structurecoordinates for a protein or a protein complex or a portion thereof, isa relative set of points that define a shape in three dimensions. Thus,it is possible that an entirely different set of coordinates coulddefine a similar or identical shape. Moreover, slight variations in theindividual coordinates will have little effect on overall shape.

The present invention includes crystals exhibiting structure coordinateswhich are similar to those set forth in Table 3 or 4 but forcrystallographic permutations of the structure coordinates,fractionalization of the structure coordinates, additions, subtractions,rotations or translations to sets of the structure coordinates or anycombinations of the above.

Alternatively, modifications in the crystal structure due to mutations,additions, substitutions, and/or deletions of amino acids, or otherchanges in any of the components that make up the crystal may alsoaccount for variations in structure coordinates. If such variations arewithin an acceptable standard error as compared to the coordinates ofTable 3 or 4, the resulting three-dimensional shape is considered to bethe same and, accordingly, the modified crystal is considered to bewithin the scope of the present invention.

Various computational analyses may be necessary to determine whether acrystal is sufficiently similar to the crystals whose structuralcoordinates are set forth in Table 3 or 4 as to be considered the same.Such analyses may be carried out in current software applications, suchas the Molecular Similarity application of QUANTA (Molecular SimulationsInc., San Diego, Calif.) version 4.1, and as described in theaccompanying User's Guide.

The Molecular Similarity application permits comparisons betweendifferent structures, different conformations of the same structure, anddifferent parts of the same structure. In general, the procedure used inMolecular Similarity to compare structures is divided into foursteps: 1) input the structures to be compared; 2) define the atomequivalences in these structures; 3) perform a fitting operation; and 4)analyze the results.

Each structure is identified by a name. One structure is identified asthe target (i.e., the fixed structure); all remaining structures areworking structures (i.e., moving structures). Since atom equivalencywithin QUANTA is defined by user input, for the purpose of thisinvention we will define equivalent atoms as protein backbone atoms (N,CA, C and O) for all conserved residues between the two structures beingcompared.

When a rigid fitting method is used, the working structure is translatedand rotated to obtain an optimum fit with the target structure. Thefitting operation uses a least squares fitting algorithm that computesthe optimum translation and rotation to be applied to the movingstructure, such that the root mean square difference of the fit over thespecified pairs of equivalent atom is an absolute minimum. This number,given in Ångströms, is reported by QUANTA.

The term “root mean square deviation” (RMSD) is a commonly known term inthe art which, in general, means the square root of the arithmetic meanof the squares of the deviations from the mean distance of correspondingatoms. It is a way to express the deviation or variation from a trend orobject.

For the purpose of this invention, any set of structure coordinates of amolecule that has a RMSD of conserved residue backbone atoms (N, CA, C,O) of less than about 2.0 Å when superimposed—using backbone atoms—onthe relevant structure coordinates of Table 3 or 4 are consideredidentical and are within the scope of the present invention. Preferablythe crystal is a modified Hdm2 protein as defined above. Preferably, theroot mean square deviation is less than about 1.5 Å, more preferablyless than 1.0 Å, even more preferably, the root mean square deviation isless than about 0.5 Å and most preferably, the root mean squaredeviation is less than about 0.1 Å.

The term “least squares” refers to a method based on the principle thatthe best estimate of a value is that in which the sum of the squares ofthe deviations of observed values is a minimum.

In one embodiment, the modified Hdm2 protein comprises the amino acidsequence of SEQ ID NO: 6. In a particular embodiment of this type, thecrystal has the space group of P2₁2₁2₁, having unit cell dimensions of:a=41.1, b=66.1, c=96.1 Angstroms. In a particular embodiment of thistype, the modified Hdm2 protein comprises the amino acid sequence of SEQID NO: 10. In a particular embodiment of this type, the crystal has thespace group of P2₁2₁2₁, having unit cell dimensions of: a=38.0, b=45.3,c=64.0 Angstroms. In a related embodiment, the crystal comprises aprotein-ligand binding complex with the modified Hdm2 protein.

In accordance with the present invention, the structure coordinates ofthe Hdm2 polypeptide and portions thereof may be stored in amachine-readable storage medium. Such data may be used for a variety ofpurposes, such as drug discovery and X-ray crystallographic analysis ofa protein crystal (e.g., for producing a three-dimensionalrepresentation of Hdm2). Accordingly, one aspect of this inventionprovides a machine-readable data storage medium comprising a datastorage material encoded with the structure coordinates set forth inTable 3 or 4. The machine-readable data storage medium may also includeany set of structure coordinates of a molecule that has a root meansquare deviation of conserved residue backbone atoms (N, CA, C, O) ofless than about 1.5 Å, preferably, less than about 1.0 Å, morepreferably less than about 0.5 Å and even more preferably less thanabout 0.1 Å when superimposed—using backbone atoms—on the relevantstructure coordinates of Table 3 or 4.

A computer system, useful in reading the machine readable data storagemedium, includes a computer comprising a central processing unit (“CPU”)and a memory storage device and is also within the scope of the presentinvention. In general, the computer system may be any computer with anoperating system such as MS-DOS, PC-DOS, Windows, OS/2, Unix, Unixvariant or MaCOS. Particularly preferred computer systems are theSilicon Graphics Octane workstation or Compaq AlphaServer DS20. Otherhardware systems and software packages will be known to those skilled inthe art.

Input hardware coupled to the computer system by input line, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be input via the use of a modem or modems connected by atelephone line or a dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. A keyboardmay also be used as an input device.

Output hardware, coupled to the computer system by output lines, maysimilarly be implemented by conventional devices. By way of example,output hardware may include a display terminal (e.g., a cathode ray tube(CRT)) for displaying a graphical representation of the threedimensional structure of Hdm2 or a portion thereof using a program suchas INSIGHT (Molecular Simulations Inc., San Diego, Calif.) or QUANTA asdescribed herein. Output hardware might also include a printer, so thathard copy output may be produced, or a disk drive, to store systemoutput for later use. In preferred embodiments, the computer possesses adisplay which is displaying a three dimensional representation of Hdm2or a fragment or homologue thereof.

In operation, the central processing unit (CPU) coordinates the use ofthe various input and output devices, coordinates data accesses frommass storage and accesses to and from working memory, and determines thesequence of data processing steps. A number of programs may be used toprocess the machine-readable data of this invention. Such programs arediscussed in reference to the computational methods of drug discovery asdescribed herein. Specific references to components of the computersystem are included as appropriate throughout the following descriptionof the data storage medium.

A magnetic data storage medium can be encoded with a machine-readabledata by a computer system as described above. Storage medium may be, forexample, a conventional floppy diskette or hard disk, having a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, on one or both sides, containing magnetic domains whosepolarity or orientation can be altered magnetically. The magneticdomains of the coating of medium may be polarized or oriented so as toencode, in a manner which may be conventional, machine readable data,such as that described herein, for execution by a system as describedherein. Storage medium may also have an opening for receiving thespindle of a disk drive or other data storage device. Alternatively, anoptically-readable data storage medium can be encoded with suchmachine-readable data, or a set of instructions. Medium can be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable.

In general, in the case of CD-ROM, as is well known, disk coating isreflective and is impressed with a plurality of pits to encode themachine-readable data. The arrangement of the pits is read by reflectinglaser light off the surface of the coating. A protective coating, whichpreferably is substantially transparent, is provided on top of thecoating.

In general, in the case of a magneto-optical disk, as is well known,disk coating has no pits, but has a plurality of magnetic domains whosepolarity or orientation can be changed magnetically when heated above acertain temperature, as by a laser. The orientation of the domains canbe read by measuring the polarization of laser light reflected from thecoating. The arrangement of the domains encodes the data as describedabove.

Structure Based Drug Design

The present invention permits the use of structure-based drug designtechniques to design, select, and synthesize chemical entities,including inhibitory compounds that are capable of binding to a Hdm2polypeptide. Also, de novo and iterative drug design methods can be usedto develop drugs from the structure of the Hdm2 crystals of thisinvention.

One particularly useful drug design technique enabled by this inventionis structure-based drug design. Structure-based drug design is a methodfor optimizing associations between a protein and a compound bydetermining and evaluating the three-dimensional structures ofsuccessive sets of protein/compound complexes.

Those skilled in the art will appreciate that association of naturalligands or substrates with the binding pockets of their correspondingreceptors, enzymes or specific binding proteins is the basis of manybiological mechanisms of action. The term “binding pocket”, as usedherein, may refer to any region of a molecule or molecular complex,that, as a result of its shape, favorably associates with anotherchemical entity or compound. Similarly, drugs may exert their biologicaleffects through association with the binding pockets of receptors and/orspecific binding proteins. Such association may occur with all or anypart of the binding pockets. An understanding of such associations willhelp lead to the design of drugs having more favorable associations withthe target protein, and thus, improved biological effects. Therefore,this information is valuable in designing potential protein inhibitors,such as inhibitors of Hdm2.

In iterative structure-based drug design, crystals of a series ofprotein/compound complexes are obtained and then the three-dimensionalstructure of each complex is solved. Such an approach provides insightinto the association between the proteins and compounds of each complex.This is accomplished by selecting compounds with inhibitory activity,obtaining crystals of a new polypeptide, solving the three-dimensionalstructure of the polypeptide, and comparing the associations between thenew protein and previously solved protein. By observing how changes inthe compound affected the protein/compound associations, theseassociations may be optimized.

In some cases, iterative structure-based drug design is carried out byforming successive protein-compound complexes and then crystallizingeach new complex. Alternatively, a pre-formed protein crystal is soakedin the presence of an inhibitor or other binding compound, therebyforming a protein/compound complex and obviating the need to crystallizeeach individual protein/compound complex. Advantageously, the modifiedHdm2 crystals provided by this invention may be soaked in the presenceof compounds, such as Hdm2 inhibitors, substrates or other ligands toprovide novel Hdm2/compound crystal complexes. In one embodiment, thecomplexes may be produced and screened using high throughput methods toquickly identify or design potential inhibitors of Hdm2.

The structure coordinates set forth in Table 3 or 4 can also be used toaid in obtaining structural information about another crystallizedmolecule or molecular complex. This may be achieved by any of a numberof well-known techniques, including molecular replacement.

The structure coordinates set forth in Table 3 or 4 can also be used fordetermining at least a portion of the three-dimensional structure ofmolecules or molecular complexes which contain at least somestructurally similar features to Hdm2. In particular, structuralinformation about another crystallized molecule or molecular complex maybe obtained by well-known techniques, including molecular replacement.

Therefore, another aspect of this invention provides a method ofutilizing molecular replacement to obtain structural information about acrystallized molecule or molecular complex, whose structure is unknown,comprising the steps of generating an X-ray diffraction pattern fromsaid crystallized molecule or molecular complex and applyingcrystallographic phases derived from at least a portion of the structurecoordinates set forth in Table 3 or 4 to the X-ray diffraction patternto generate a three-dimensional electron density map of the molecule ormolecular complex whose structure is unknown.

Once the structure coordinates of a protein crystal have beendetermined, they are useful in solving the structures of other crystals.In addition, the structure of Hdm2 homologues may be determined from thestructural coordinates of the present invention. For example,polypeptides may be crystallized and their structures elucidated by, forexample, difference Fourier techniques and molecular replacement.

By using molecular replacement, all or part of the structure coordinatesof the Hdm2 polypeptide provided by this invention (and set forth inTable 3 or 4) can be used to determine the previously unknown structureof a crystallized molecule or molecular complex more quickly andefficiently than attempting to determine such information ab initio.

Molecular replacement provides an accurate estimation of the phases foran unknown structure. Phases are a factor in equations used to solvecrystal structures that cannot be measured experimentally. Obtainingaccurate values for the phases, by methods other than molecularreplacement, is a time-consuming process. However, when the crystalstructure of a protein containing a homologous portion has been solved,the phases from the known structure may provide a satisfactory estimateof the phases for the unknown structure.

Thus, this method involves generating a preliminary model of a moleculeor molecular complex whose structure coordinates are unknown, byorienting and positioning the relevant portion of the modified Hdm2crystal according to Table 3 or 4 within the unit cell of the crystal ofthe unknown molecule or molecular complex so as best to account for theobserved X-ray diffraction pattern amplitudes to generate an electiondensity map of the structure whose coordinates are unknown. This, inturn, can be subjected to any well-known model building and structurerefinement techniques to provide a final, accurate structure of theunknown crystallized molecule or molecular complex (Lattman, “Use of theRotation and Translation Functions”, in Meth. Enzymol., 115: 55-77(1985); Rossman, ed., “The Molecular Replacement Method”, Int. Sci. Rev.Ser., No. 13, Gordon & Breach, New York (1972)).

Phase information from the structure coordinates of the presentinvention may be used to elucidate the structure of other crystals. Forexample, the structure of Hdm2 in complex with other atoms or moleculesmay be elucidated. Such complexes include, for example, those containingatoms soaked into or cocrystallized within the crystal lattice. Otherstructures which can be elucidated using the phase information of thepresent invention include for example other proteases or homologues ormutants thereof having sufficient three-dimensional structure similarityto Hdm2 complex as to be solved using molecular replacement. Also, theseprotein molecules in a complex with a small molecule substrate(s),inhibitor(s), transition state analog(s), product(s) or analog(s) of anyof these may also be solved using the phase information of the presentinvention. Other complexes whose structure can be elucidated from thephase information of the present invention include a modified Hdm2complexed with an inhibitor other than those presented herein. Complexescontaining a combination of the above molecules may also be solved usingthe phase information of the present invention.

The structure of any portion of any crystallized molecule or molecularcomplex that is sufficiently homologous to any portion of the modifiedHdm2 protein can be solved by this method. The difference Fourier methodsimply calculates an electron density map using phases calculated fromthe structure coordinates and observed diffraction amplitudes from acrystal of an unknown structure. This method is often used to solvestructures of protein/ligand complexes where the ligand is small anddoes not affect the crystal form significantly.

In a preferred embodiment, the method of molecular replacement isutilized to obtain structural information about a molecule wherein themolecule comprises a Hdm2 polypeptide complex. The structure coordinatesof modified Hdm2 provided by this invention are particularly useful insolving the structure of other crystal forms of Hdm2 polypeptidecomplexes. This approach enables the determination of the optimal sitesfor interaction between chemical entities, including interaction ofcandidate inhibitors with Hdm2.

Modified Hdm2 crystals may be studied using well-known X-ray diffractiontechniques and may be refined versus X-ray data to 3 Å resolution orbetter to an R_(free) value of about 0.40 or less using computersoftware such as X-PLOR (Yale University, 1992, distributed by MolecularSimulations, Inc.; see e.g., Blundell & Johnson, supra; Meth. Enzymol.,vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985)). Thisinformation may be used to optimize known Hdm2 inhibitors and to designnew Hdm2 inhibitors.

Once a three-dimensional structure of a crystal comprising a modifiedHdm2 protein in a protein-ligand complex is determined, the potentialinhibitor of Hdm2 can be examined through the use of computer modelingusing a docking program such as GRAM, DOCK, or AUTODOCK [Dunbrack etal., Folding & Design, 2:27-42 (1997)]. This procedure can includecomputer fitting of potential inhibitors to the modified Hdm2 protein toascertain how well the shape and the chemical structure of the potentialmodulator will interact with the modified Hdm2 protein [Bugg et al.,Scientific American, December:92-98 (1993); West et al., TIBS, 16:67-74(1995)]. Computer programs can also be employed to estimate theattraction, repulsion, and steric hindrance of the modified Hdm2 proteinwith an inhibitor.

Generally the tighter the fit, the lower the steric hindrances, and thegreater the attractive forces, the more potent the inhibitor, sincethese properties are consistent with a tighter binding constant.Furthermore, the more specificity in the design of a potential drug themore likely that the drug will not interact as well with other proteins.This will minimize potential side-effects due to unwanted interactionswith other proteins.

Compounds that may be used initially have been discussed by Chene[Nature 3:102-109 (2003)]. In addition, the present invention disclosesthe acetylated tripeptides, (a) Ac-^(6Cl)WAC_(3c)E and (b)Ac-^(6Br)WAC_(3c)E, as shown respectively below, which can individuallybind a modified Hmd2 protein in protein-ligand complex and form an X-raydiffractable crystal.

These ligands then can be systematically modified by computer modelingprograms until one or more promising potential analogs are identified.Such analysis has been shown to be effective in the development of HIVprotease inhibitors [Lam et al., Science 263:380-384 (1994); Wlodawer etal., Ann. Rev. Biochem. 62:543-585 (1993); Appelt, Perspectives in DrugDiscovery and Design 1:23-48 (1993); Erickson, Perspectives in DrugDiscovery and Design 1:109-128 (1993)]. Alternatively, a potentialinhibitor initially can be obtained by screening a random peptidelibrary or a chemical library. In the former case, a random peptidelibrary can be produced by recombinant bacteriophage, for example,[Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc.Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science,249:404-406 (1990)]. This approach may be particularly useful in thiscase since the natural binding partner for Mdm2 is the p53 protein. Inany case, a peptide selected in this manner could then be systematicallymodified by computer modeling programs, as described above.

If a potential inhibitor is a small organic compound, it can be selectedfrom a library of chemicals, as are commercially available.Alternatively, the small organic compound may be synthesized de novo.Once obtained, the potential inhibitor can be further tested in astandard binding and/or functional assay with Hdm2, a modified Hdm2protein or active fragments thereof.

For example, a binding assay can be performed following the attachmentof the Hdm2 protein to a solid support. Methods for placing Hdm2 proteinon the solid support are well known in the art and include such thingsas linking biotin to the Hdm2 protein and linking avidin to the solidsupport. The solid support can be washed to remove unbound protein. Asolution of a labeled potential inhibitor can be contacted with thesolid support. The solid support is washed again to remove the potentialinhibitor not bound to the support. The amount of labeled potentialinhibitor remaining with the solid support, and thereby bound to theHdm2 protein can be determined. Alternatively, or in addition, thedissociation constant between the labeled potential inhibitor and theHdm2 protein, for example, can be determined. Suitable labels for eitherthe Hdm2 protein or the potential inhibitor include, radioactive labels(e.g., ¹⁴C, ¹H,) and fluorescent labels such as fluoresceinisothiocyanate (FITC).

In another embodiment, a Biacore machine can be used to determine thebinding constant of the Hdm2 protein with a potential inhibitor[O'Shannessy et al. Anal. Biochem. 212:457-468 (1993); Schuster et al.,Nature 365:343-347 (1993)].

In addition, an inhibitor can be identified using an ELISA-basedcompetition assay [Bottger et al., Oncogene 13:2141-2147 (1996); Bottgeret al., J. Miol. Biol. 269:744-756 (1997)]. For example, the p53 proteinor Hdm2-binding fragment thereof can be biotinylated and immobilized ona streptavidin-coated ELISA plate. Hdm2 is then incubated alone (in acontrol) or in the presence of a potential inhibitor. The Hdm2 solutionsare then individually contacted with the immobilized p53 protein orHdm2-binding fragment thereof. The binding of the Hdm2 is thendetermined, e.g., with a detectable anti-Hdm2 antibody. When the amountof Hdm2 detected is lower in the sample incubated with the potentialinhibitor relative to the control, the potential inhibitor is identifiedas an inhibitor.

When a promising inhibitor is identified, a crystal comprising aprotein-ligand complex of the inhibitor and a modified Hdm2 protein canbe prepared. The three-dimensional structure of the resultingcrystalline protein-ligand complex can then be determined by molecularreplacement analysis, for example.

Molecular replacement involves the use of a known three-dimensionalstructure as a search model to determine the structure of a closelyrelated molecule or protein-ligand complex in a different crystallineform. The measured X-ray diffraction properties of the new crystal arecompared with the search model structure to compute the position andorientation of the protein in the new crystal. Computer programs thatcan be used include: X-PLOR [Brunger et al., Acta Crystallogr. A46:585-593 (1990); Brunger et al., Acta Crystallogr. D BiolCrystallogr., 54:905-921 (1998)], CNS, (Crystallography and NMR System,a next level of XPLOR), and AMORE [Navaza, Acta Crystallographics ASO,157-163 (1994)]. Once the position and orientation are known, anelectron density map can be calculated using the search model to provideX-ray phases. Thereafter, the electron density is inspected forstructural differences and the search model is modified to conform tothe new structure. Using this approach, it is possible to solve thethree-dimensional structures of crystals of any protein-ligand complexof the modified Hdm2 protein.

For all of the drug screening assays described herein, furtherrefinements to the structure of the drug will generally be necessary andcan be made by the successive iterations of any and/or all of the stepsprovided by the particular drug screening assay and/or in combinationwith other such drug screening assays.

A candidate drug selected by performing structure based drug design canthen be assayed in situ and/or in vivo. For example, a candidate drugcan be evaluated for cellular activity by incubating the candidate drugin cell cultures, e.g. using HCT-116 cells or OSA-CL cells, and thenmeasuring its effect on cellular proliferation and expression levels ofproteins that are transcriptionally regulated by p53 such as p21waf1 andHdm2 [Chene et al., J. Mol. Biol. 299:245-256 (2000)]. A candidate drugis identified as a drug if in the presence of the drug relative to inits absence, the amount of cellular proliferation decreases and/or theamount of a protein that is transcriptionally regulated by p53increases.

Indeed, methods of testing such potential candidate drugs in animalmodels are well known in the art. The potential drugs can beadministered by a variety of ways including topically, orally,subcutaneously, or intraperitoneally depending on the proposed use.Generally, at least two groups of animals are used in the assay, with atleast one group being a control group that is administered theadministration vehicle without the potential drug.

TABLE 2 TABLE OF SEQUENCES SEQ ID NO: Type Description 1 N.A. WT Hdm2(17-125) 2 A.A. WT Hdm2 (17-125) 3 N.A. modified Hdm2 (X₁-X₇) 4 A.A.modified Hdm2 (X₁-X₇) 5 N.A. modified Hdm2 (Y76H) 6 A.A. modified Hdm2(Y76H) 7 N.A. modified Hdm2 (F55Y) 8 A.A. modified Hdm2 (F55Y) 9 N.A.modified Hdm2 (F55Y/Y76H) 10 A.A. modified Hdm2 (F55Y/Y76H) 11 N.A.modified Hdm2 (HK₅) 12 A.A. modified Hdm2 (HK₅) 13 N.A. Hdm2 (F55Y)Primer 14 N.A. RChdm2 (F55Y) Primer 15 N.A. Hdm2 (Y76H) Primer 16 N.A.RChdm2 (Y76H) 17 N.A. Y104S-GTAILOR-F Primer 18 N.A. Y104-GTAILOR-RPrimer

The present invention may be better understood by reference to thefollowing non-limiting examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate certain embodiments of the invention. They should in no waybe construed as limiting the broad scope of the invention.

EXAMPLES Example 1 Preparation of Modified Hdm2

Preparation of Hdm2 Constructs

Hdm2 was either modified with a single amino acid change, or with adouble amino acid change using the QuickChange kit (Stratagene, LaJolla, Calif., USA) and the pET32-Xa/LIC-hdm2 (17-125) vector as atemplate. The pET32-Xa/LIC parental vector was obtained from Novagen(San Diego, Calif.). Hdm2(F55Y), Hdm2(Y76H) and Hdm2(F55Y/Y76H) wereconstructed in this manner.

A modified Hdm2 having three amino acid substitutions,Hdm2(F55Y/Y76H/Y104S) was generated using GENETAILOR Site-directedMutagenesis system (Invitrogen, Carlsbad, Calif., USA) with the vectorindicated above as the template. The following primers were used togenerate the above-identified modified Hdm2 proteins:

(1) Hdm2(F55Y) Primer: SEQ ID NO:13 5′CTATGAAAGAGGTTCTTTATTATCTTGGCCAGTATATTATGAC 3′ (2) RChdm2(F55Y) Primer:SEQ ID NO:14 5′ GTCATAATATACTGGCCAAGATAATAAAGAACCTCTTTCATAG 3′ (3)Hdm2(Y76H) Primer: SEQ ID NO:15 5′GAGAAGCAACAACATATTGTACATTGTTCAAATGATCTTCTAGG 3′ (4) RChdm2(Y76H) Primer:SEQ ID NO:16 5′ CCTAGAAGATCATTTGAACAATGTACAATATGTTGTTGCTTCTC 3′ (5)Y104S-GTAILOR-F Primer: SEQ ID NO:17 5′ CAGGAACTTGGTAGTAGTCAATCAGCAGG 3′(6) Y104-GTAILOR-R Primer: SEQ ID NO:18 5′GACTACTACCAAGTTCCTGGAGATCATGGT 3′

QuickChange mutagenesis was performed in two steps as previouslydescribed [Wang et al., BioTechniques 26:680-682(1999)]. In the firststage two extension reactions were performed in separate tubes; onecontaining the forward primer and the other containing the reverseprimer. After two cycles, the two reactions were mixed and the standardQuickChange mutagenesis procedure was carried out for an additional 18cycles. Following amplification, the parental strand was digested with1U of Dpn1 for 2 hours and an aliquot was transformed into DH5-α cells.The GENETAILOR mutagenesis was performed according to the manufacturer'sinstructions except that pfu turbo DNA polymerase was used instead ofthe polymerase recommended by manufacture. All constructs were confirmedby sequencing (GeneWiz, New York, N.Y.).

Expression of Modified Hdm2

A colony from freshly transformed cells was grown at 37° C. to anoptical density (OD) of 2.0 in 10 ml TERRIFIC broth (Mediatech, Inc.)containing 100 μg/ml carbenicilin and 1% glucose. This 10 ml culture wasthen used to inoculate a 1 liter culture having the same mediumcomposition. The 1 liter culture was grown at 37° C. to an OD of 2.0,stored at 4° C. overnight, and then used to inoculate a 10 liter tankcontaining TERRIFIC broth and 100 μg/ml carbenicilin. The 10 literculture was grown at 37° C. to an OD of 1.5-2.0 before lowering thetemperature to 16° C. The 10 liter culture was then induced with 1 mMIPTG, and the cells were harvested 18 hours post-induction.

Purification of Modified Hdm-2 Proteins

The purification protocol as exemplified herein for Hdm2(F55Y/Y76H) isapplicable for all of the modified Hdm2 proteins of the presentinvention.

IPTG-induced cells containing Hdm2(F55Y/Y76H) were harvested from the 10liter fermentation, as described above. The cells were suspended in 500ml of 50 mM Tris-Cl Buffer, pH 8.0_(rt) (room temperature), 0.3 M NaCl,10% (v/v) glycerol, 5 mM β-mercaptoethanol, 25 mM imidazole, 18,000Units/liter endonuclease (ultrapure benzonase; SIGMA), and 6 ml/liter ofCALBIOCHEM Protease Inhibitor Cocktail III. (All processing wasperformed at 4° C.) To homogenize the resulting cell suspension, it waspassed through a large OMNI Mixer probe for 45 seconds, three times. Thecell suspension was kept on ice for 2 minutes between each 45 secondpassage. The cells were then broken by three passages of the homogenizedcell suspension through a Microfluidizer. The extract was recovered bycentrifugation at 205,000×g for 80 minutes at 4° C.

The resulting 645 ml extract was mixed end over end for 50 minutes with28 ml of QIAGEN Ni-NTA SUPERFLOW resin, which had been equilibrated withthe equilibration buffer [50 mM Tris-Cl, pH 8.0_(rt), 0.3 M NaCl, 5 mMβ-mercaptoethanol and 25 mM imidazole]. The supernatant was decanted,and the resin was then washed with 600 ml of the equilibration buffer.The resin was poured into a 2.6×5.3 cm column, washed with an additional200 ml of equilibration buffer at 3.6 ml/min, and finally eluted with 50mM Tris-Cl, pH 8.0_(rt), 0.1 M NaCl, 250 mM imidazole, 5 mMβ-mercaptoethanol and 20% glycerol.

A 0.64 ml volume of 0.5 M CaCl₂ was added to the eluted fusion proteinpool (195 mg of protein in 63 ml of elution buffer). The pooled proteinwas then diluted to 1 mg/ml with 50 mM Tris-Cl, pH 8.0_(rt), 0.1 M NaCl,10% glycerol, 5 mM CaCl₂ and 5 mM β-mercaptoethanol. A 1.95 ml volume of2000 Units/ml Factor Xa protease (NOVAGEN) was added, and the pooledprotein was dialyzed overnight versus 3.87 liters of the same buffer.

A 4.33 ml volume of 1 M imidazole, pH 8.0, was added to the 200 ml ofcleaved, pooled fusion protein, to bring the imidazole concentration to24 mM. The pooled protein was then applied at a rate of 3.6 m/min to a50 ml (2.6×9.4 cm) column of QIAGEN Ni-NTA SUPERFLOW resin that had beenequilibrated with equilibration buffer. The column was then washed withthe equilibration buffer.

The 230 ml flow-through was dialyzed versus three changes (6 liters, 5liters and 5 liters) of Buffer A [25 mM Hepes-KOH, pH 7.5, 0.15 M KCl, 1mM Na₂-EDTA, 0.03% sodium azide and 5 mM dithiothreitol]. Allmanipulations of the modified Hdm2 were in Buffer A from this point on.The dialyzed pooled protein was concentrated to 8.5 ml with an AMICONYM10 membrane, centrifuged at 205,000×g for 15 minutes, and then appliedto a 2.6×60 cm column of PHARMACIA SUPERDEX-75 at a flow rate of 0.8ml/min. The resulting eluant was collected in 3.2 ml fractions.

Fractions 66-75 contained the purified, modified Hdm2 monomer. Thesefractions were pooled and a protein concentration of 3.6 mg/ml wasdetermined using ε₂₇₆=10,150 M⁻¹ cm⁻¹ in 20 mM sodium phosphate, pH 6.5,with 6 M guanidine hydrochloride (ExPASy-ProtParam Tool). Thisdetermination correlated with that determined by the Bradford dyebinding assay (BIORAD), using bovine serum albumin as the proteinstandard.

EXAMPLE 2 Preparation and Crystallization of Hdm2 F55Y/Y76H-TripeptideComplex

The modified Hdm2 (F55Y/Y76H) protein was prepared by means ofQuickChange mutagenesis as disclosed in Example 1 using the appropriateprimers. A small scale expression study was carried out to evaluate thesolubility and expression level of this construct. The expression levelof the soluble Hdm2(F55Y/Y76H) protein is 2 to 3 fold higher than wildtype. 110 mgs of the Hdm2(F55Y/Y76H) was purified from a 10 L culturethrough the four-step purification protocol described above.

A 13 ml aliquot of the 3.6 mg/ml pool was concentrated to a 1.25 mMconcentration (in 3 ml), on an AMICON 5000 mwco Ultrafree membrane. A 3ml aliquot of 8.5 mM Ac-^(6Cl)WAC_(3c)E (MW=535) in Buffer A was addedto the concentrated aliquot. The complex was incubated at roomtemperature for 10 minutes, and then concentrated as above, to a finalvolume of 1.3 ml.

The HDM2(F55Y/Y76H) protein-tripeptide complex was crystallized using ahanging-drop vapor diffusion method. The protein (0.5 μl; 34 mg/ml) in25 mM Hepes-potassium hydroxide, pH7.5, 0.15 M potassium chloride, 1 mMEDTA, 0.03% sodium azide and 5 mM DTT buffer was mixed with an equalvolume of precipitant solution [1.4 M tri sodium citrate, 0.1 M sodiumHepes, pH 7.5] placed on the underside of a siliconized glass coverslipand sealed in close proximity to 1 ml of the precipitant solution.Crystallization plates were incubated at 22° C.; rectangular rodcrystals (0.02×0.2 mm) grew over 2-30 days.

Prior to data collection, crystals were washed with the reservoirsolution of the crystallization setup and transferred into the samesolution with 20% glycerol added. The crystals were then flash-cooled ina nitrogen stream at 95 K. X-ray diffraction was collected using aRigaku generator equipped with a Raxis 4++ detector. Data wereintegrated and scaled using the HKL package.

Data Collection Statistics:

Resolution 50.0-1.70 Å No. of collected reflections 135908 No. of uniquereflections (F >= 0)  12484 R-sym  6.7% Percent of theoretical(I/s >= 1) 98.7% Unit Cell a = 37.999 Å, b = 45.333 Å, c = 63.999 Å, α =β = γ = 90° Space Group P2₁2₁2₁ Asymmetric unit 1 molecule

The crystal structure was solved using molecular replacement using thesearch models 1YCQ and 1YCR from the PDB. Refinement was done using theprogram CNX.

Theoretical Number of Reflections 9121

Resolution Limits 50.0-1.90 Å Number of unobserved reflections  106(1.2%) Number of reflections in working set 8592 (98.8%) Number ofreflections in test set  423 (4.6%) Number of protein residues 87 Numberof solvent atoms 44 R-factor  0.223 R-free  0.243 RMSD bond length    0.0083 Å RMSD bond angles 1.46°

The structural coordinates for the above-described Hdm2 crystal are setforth below in Table 3, which is in Protein Data Bank (PDB) file format.The numbered columns refer to the following:

Col. # Reference 1 Atomic coordinate records for standard groups 2 Atomserial number 3 Atom name 4 Residue name 5 Residue sequence number 6Orthogonal coordinates for X in Angstroms 7 Orthogonal coordinates for Yin Angstroms 8 Orthogonal coordinates for Z in Angstroms 9 Occupancy 10Temperature factor 11 Element symbol

TABLE 3 1 2 3 4 5 6 7 8 9 10 11 ATOM 1 CB GLU 25 8.599 29.031 2.725 1.0052.10 C ATOM 2 CG GLU 25 7.336 29.192 3.556 1.00 55.02 C ATOM 3 CD GLU25 6.648 27.844 3.697 1.00 57.01 C ATOM 4 OE1 GLU 25 5.517 27.697 3.1861.00 58.12 O ATOM 5 OE2 GLU 25 7.235 26.932 4.322 1.00 57.62 O ATOM 6 CGLU 25 10.683 30.035 1.753 1.00 47.48 C ATOM 7 O GLU 25 10.654 29.6820.574 1.00 48.97 O ATOM 8 N GLU 25 8.564 31.353 1.814 1.00 50.30 N ATOM9 CA GLU 25 9.389 30.323 2.511 1.00 49.61 C ATOM 10 N THR 26 11.81630.195 2.431 1.00 43.07 N ATOM 11 CA THR 26 13.119 29.941 1.824 1.0037.71 C ATOM 12 CB THR 26 14.267 30.244 2.807 1.00 38.14 C ATOM 13 OG1THR 26 14.227 31.628 3.174 1.00 38.51 O ATOM 14 CG2 THR 26 15.613 29.9422.163 1.00 37.88 C ATOM 15 C THR 26 13.216 28.482 1.385 1.00 34.76 CATOM 16 O THR 26 12.823 27.579 2.120 1.00 34.28 O ATOM 17 N LEU 2713.730 28.261 0.179 1.00 29.30 N ATOM 18 CA LEU 27 13.872 26.915 −0.3651.00 28.75 C ATOM 19 CB LEU 27 13.479 26.866 −1.842 1.00 28.72 C ATOM 20CG LEU 27 12.043 27.255 −2.190 1.00 29.98 C ATOM 21 CD1 LEU 27 11.82327.208 −3.696 1.00 30.85 C ATOM 22 CD2 LEU 27 11.037 26.358 −1.482 1.0030.07 C ATOM 23 C LEU 27 15.240 26.271 −0.161 1.00 26.20 C ATOM 24 O LEU27 16.270 26.922 −0.316 1.00 26.92 O ATOM 25 N VAL 28 15.234 24.9890.196 1.00 24.29 N ATOM 26 CA VAL 28 16.465 24.230 0.402 1.00 22.04 CATOM 27 CB VAL 28 16.566 23.684 1.845 1.00 21.66 C ATOM 28 CG1 VAL 2816.716 24.839 2.822 1.00 20.57 C ATOM 29 CG2 VAL 28 15.337 22.849 2.1801.00 18.74 C ATOM 30 C VAL 28 16.549 23.066 −0.585 1.00 22.51 C ATOM 31O VAL 28 15.526 22.500 −0.979 1.00 20.60 O ATOM 32 N ARG 29 17.77022.733 −0.996 1.00 21.42 N ATOM 33 CA ARG 29 18.010 21.641 −1.937 1.0023.64 C ATOM 34 CB ARG 29 18.869 22.116 −3.107 1.00 27.62 C ATOM 35 CGARG 29 19.162 21.053 −4.151 1.00 33.83 C ATOM 36 CD ARG 29 20.043 21.611−5.267 1.00 38.61 C ATOM 37 NE ARG 29 19.435 22.740 −5.965 1.00 42.13 NATOM 38 CZ ARG 29 18.360 22.653 −6.742 1.00 45.54 C ATOM 39 NH1 ARG 2917.763 21.483 −6.929 1.00 47.02 N ATOM 40 NH2 ARG 29 17.882 23.740−7.337 1.00 46.93 N ATOM 41 C ARG 29 18.696 20.470 −1.227 1.00 20.05 CATOM 42 O ARG 29 19.851 20.573 −0.819 1.00 18.84 O ATOM 43 N PRO 3017.988 19.343 −1.078 1.00 19.58 N ATOM 44 CD PRO 30 16.511 19.312 −1.0311.00 21.57 C ATOM 45 CA PRO 30 18.498 18.137 −0.425 1.00 20.02 C ATOM 46CB PRO 30 17.278 17.234 −0.444 1.00 22.34 C ATOM 47 CG PRO 30 16.23418.184 0.016 1.00 21.93 C ATOM 48 C PRO 30 19.678 17.493 −1.160 1.0019.19 C ATOM 49 O PRO 30 19.624 17.323 −2.379 1.00 18.14 O ATOM 50 N LYS31 20.737 17.144 −0.433 1.00 17.57 N ATOM 51 CA LYS 31 21.870 16.450−1.044 1.00 17.54 C ATOM 52 CB LYS 31 23.083 16.416 −0.110 1.00 17.17 CATOM 53 CG LYS 31 23.588 17.814 0.242 1.00 21.31 C ATOM 54 CD LYS 3124.792 17.784 1.175 1.00 24.34 C ATOM 55 CE LYS 31 25.250 19.198 1.4991.00 27.70 C ATOM 56 NZ LYS 31 26.424 19.216 2.411 1.00 30.99 N ATOM 57C LYS 31 21.367 15.049 −1.436 1.00 18.76 C ATOM 58 O LYS 31 20.31614.612 −0.961 1.00 17.24 O ATOM 59 N PRO 32 22.093 14.338 −2.319 1.0018.30 N ATOM 60 CD PRO 32 23.329 14.791 −2.991 1.00 21.43 C ATOM 61 CAPRO 32 21.764 13.000 −2.820 1.00 19.62 C ATOM 62 CB PRO 32 23.111 12.503−3.293 1.00 22.05 C ATOM 63 CG PRO 32 23.536 13.680 −4.080 1.00 21.40 CATOM 64 C PRO 32 21.004 11.990 −1.947 1.00 19.34 C ATOM 65 O PRO 3219.883 11.591 −2.299 1.00 17.19 O ATOM 66 N LEU 33 21.594 11.559 −0.8331.00 18.45 N ATOM 67 CA LEU 33 20.911 10.581 0.017 1.00 18.88 C ATOM 68CB LEU 33 21.788 10.020 1.142 1.00 21.73 C ATOM 69 CG LEU 33 23.0509.209 0.871 1.00 28.04 C ATOM 70 CD1 LEU 33 24.067 9.989 0.052 1.0029.17 C ATOM 71 CD2 LEU 33 23.663 8.738 2.191 1.00 29.05 C ATOM 72 C LEU33 19.597 11.062 0.602 1.00 17.92 C ATOM 73 O LEU 33 18.603 10.340 0.5651.00 16.48 O ATOM 74 N LEU 34 19.588 12.277 1.140 1.00 17.19 N ATOM 75CA LEU 34 18.365 12.817 1.719 1.00 15.57 C ATOM 76 CB LEU 34 18.63214.142 2.431 1.00 17.51 C ATOM 77 CG LEU 34 17.436 14.863 3.052 1.0018.19 C ATOM 78 CD1 LEU 34 16.656 13.966 4.007 1.00 16.62 C ATOM 79 CD2LEU 34 17.885 16.147 3.738 1.00 16.73 C ATOM 80 C LEU 34 17.316 12.9950.638 1.00 17.16 C ATOM 81 O LEU 34 16.128 12.769 0.870 1.00 16.85 OATOM 82 N LEU 35 17.755 13.385 −0.553 1.00 15.69 N ATOM 83 CA LEU 3516.814 13.578 −1.644 1.00 18.62 C ATOM 84 CB LEU 35 17.508 14.148 −2.8851.00 20.48 C ATOM 85 CG LEU 35 16.605 14.406 −4.097 1.00 23.23 C ATOM 86CD1 LEU 35 15.470 15.356 −3.733 1.00 23.45 C ATOM 87 CD2 LEU 35 17.38914.944 −5.293 1.00 23.24 C ATOM 88 C LEU 35 16.160 12.238 −1.966 1.0019.55 C ATOM 89 O LEU 35 14.951 12.155 −2.195 1.00 17.74 O ATOM 90 N LYS36 16.964 11.181 −1.955 1.00 18.27 N ATOM 91 CA LYS 36 16.453 9.859−2.271 1.00 22.37 C ATOM 92 CB LYS 36 17.610 8.864 −2.413 1.00 24.10 CATOM 93 CG LYS 36 17.212 7.444 −2.785 1.00 30.65 C ATOM 94 CD LYS 3618.461 6.576 −2.920 1.00 32.62 C ATOM 95 CE LYS 36 18.128 5.145 −3.3071.00 36.89 C ATOM 96 NZ LYS 36 19.362 4.312 −3.442 1.00 37.42 N ATOM 97C LYS 36 15.413 9.413 −1.246 1.00 22.40 C ATOM 98 O LYS 36 14.435 8.751−1.597 1.00 22.93 O ATOM 99 N LEU 37 15.605 9.802 0.013 1.00 22.25 NATOM 100 CA LEU 37 14.659 9.432 1.065 1.00 21.53 C ATOM 101 CB LEU 3715.231 9.702 2.453 1.00 24.79 C ATOM 102 CG LEU 37 16.511 8.980 2.8651.00 27.14 C ATOM 103 CD1 LEU 37 16.996 9.454 4.226 1.00 29.28 C ATOM104 CD2 LEU 37 16.317 7.474 2.855 1.00 29.24 C ATOM 105 C LEU 37 13.35510.197 0.894 1.00 22.46 C ATOM 106 O LEU 37 12.269 9.636 1.053 1.0019.80 O ATOM 107 N LEU 38 13.470 11.479 0.557 1.00 21.33 N ATOM 108 CALEU 38 12.296 12.322 0.369 1.00 21.81 C ATOM 109 CB LEU 38 12.693 13.7940.252 1.00 22.60 C ATOM 110 CG LEU 38 13.394 14.398 1.467 1.00 23.43 CATOM 111 CD1 LEU 38 13.905 15.806 1.185 1.00 24.29 C ATOM 112 CD2 LEU 3812.491 14.362 2.685 1.00 24.94 C ATOM 113 C LEU 38 11.483 11.884 −0.8361.00 24.00 C ATOM 114 O LEU 38 10.254 11.986 −0.830 1.00 25.09 O ATOM115 N LYS 39 12.160 11.381 −1.865 1.00 24.12 N ATOM 116 CA LYS 39 11.44210.928 −3.045 1.00 28.66 C ATOM 117 CB LYS 39 12.339 10.855 −4.282 1.0029.59 C ATOM 118 CG LYS 39 12.939 12.179 −4.728 1.00 30.58 C ATOM 119 CDLYS 39 13.798 11.969 −5.968 1.00 34.21 C ATOM 120 CE LYS 39 14.41413.266 −6.459 1.00 35.99 C ATOM 121 NZ LYS 39 13.376 14.268 −6.818 1.0037.93 N ATOM 122 C LYS 39 10.766 9.588 −2.803 1.00 29.02 C ATOM 123 OLYS 39 9.784 9.256 −3.468 1.00 30.57 O ATOM 124 N SER 40 11.270 8.823−1.839 1.00 27.39 N ATOM 125 CA SER 40 10.671 7.526 −1.557 1.00 27.21 CATOM 126 CB SER 40 11.619 6.625 −0.768 1.00 29.11 C ATOM 127 OG SER 4011.934 7.200 0.484 1.00 31.61 O ATOM 128 C SER 40 9.371 7.739 −0.7911.00 26.87 C ATOM 129 O SER 40 8.522 6.850 −0.731 1.00 24.88 O ATOM 130N VAL 41 9.217 8.931 −0.222 1.00 23.16 N ATOM 131 CA VAL 41 8.020 9.2650.534 1.00 24.52 C ATOM 132 CB VAL 41 8.321 10.392 1.553 1.00 25.80 CATOM 133 CG1 VAL 41 7.053 10.879 2.197 1.00 27.99 C ATOM 134 CG2 VAL 419.271 9.873 2.620 1.00 25.81 C ATOM 135 C VAL 41 6.933 9.718 −0.436 1.0024.07 C ATOM 136 O VAL 41 5.750 9.780 −0.083 1.00 22.91 O ATOM 137 N GLY42 7.343 10.018 −1.665 1.00 23.67 N ATOM 138 CA GLY 42 6.394 10.446−2.678 1.00 23.84 C ATOM 139 C GLY 42 6.685 11.764 −3.373 1.00 22.51 CATOM 140 O GLY 42 6.165 12.010 −4.461 1.00 21.84 O ATOM 141 N ALA 437.508 12.616 −2.767 1.00 22.69 N ATOM 142 CA ALA 43 7.817 13.916 −3.3611.00 23.42 C ATOM 143 CB ALA 43 8.331 14.868 −2.287 1.00 26.17 C ATOM144 C ALA 43 8.830 13.811 −4.497 1.00 23.99 C ATOM 145 O ALA 43 9.99613.496 −4.270 1.00 23.65 O ATOM 146 N GLN 44 8.369 14.091 −5.715 1.0023.66 N ATOM 147 CA GLN 44 9.194 14.028 −6.925 1.00 26.00 C ATOM 148 CBGLN 44 8.416 13.405 −8.091 1.00 27.49 C ATOM 149 CG GLN 44 7.957 11.970−7.853 1.00 29.74 C ATOM 150 CD GLN 44 9.160 11.066 −7.633 1.00 33.73 CATOM 151 OE1 GLN 44 10.295 11.436 −7.932 1.00 36.21 O ATOM 152 NE2 GLN44 8.915 9.878 −7.092 1.00 35.39 N ATOM 153 C GLN 44 9.814 15.363 −7.3441.00 28.84 C ATOM 154 O GLN 44 9.895 15.669 −8.532 1.00 33.20 O ATOM 155N LYS 45 10.239 16.162 −6.373 1.00 26.37 N ATOM 156 CA LYS 45 10.84917.453 −6.676 1.00 26.53 C ATOM 157 CB LYS 45 9.937 18.606 −6.267 1.0026.39 C ATOM 158 CG LYS 45 9.609 18.638 −4.794 1.00 26.84 C ATOM 159 CDLYS 45 8.700 19.806 −4.469 1.00 28.76 C ATOM 160 CE LYS 45 8.370 19.843−2.986 1.00 27.65 C ATOM 161 NZ LYS 45 7.477 20.988 −2.669 1.00 30.55 NATOM 162 C LYS 45 12.229 17.574 −6.037 1.00 25.49 C ATOM 163 O LYS 4512.575 16.786 −5.161 1.00 24.24 O ATOM 164 N ASP 46 13.020 18.548 −6.4801.00 26.71 N ATOM 165 CA ASP 46 14.360 18.726 −5.925 1.00 28.66 C ATOM166 CB ASP 46 15.403 18.827 −7.040 1.00 35.21 C ATOM 167 CG ASP 4615.144 19.991 −7.980 1.00 40.54 C ATOM 168 OD1 ASP 46 14.152 20.724−7.771 1.00 42.44 O ATOM 169 OD2 ASP 46 15.936 20.172 −8.932 1.00 45.73O ATOM 170 C ASP 46 14.534 19.865 −4.913 1.00 25.87 C ATOM 171 O ASP 4615.587 19.975 −4.286 1.00 24.85 O ATOM 172 N THR 47 13.513 20.703 −4.7471.00 23.77 N ATOM 173 CA THR 47 13.586 21.811 −3.788 1.00 23.33 C ATOM174 CB THR 47 13.618 23.193 −4.476 1.00 25.02 C ATOM 175 OG1 THR 4712.413 23.394 −5.219 1.00 27.77 O ATOM 176 CG2 THR 47 14.814 23.291−5.398 1.00 26.67 C ATOM 177 C THR 47 12.398 21.764 −2.843 1.00 21.70 CATOM 178 O THR 47 11.266 21.522 −3.263 1.00 21.88 O ATOM 179 N TYR 4812.670 21.995 −1.563 1.00 19.26 N ATOM 180 CA TYR 48 11.649 21.927−0.524 1.00 19.39 C ATOM 181 CB TYR 48 11.819 20.647 0.286 1.00 20.20 CATOM 182 CG TYR 48 11.751 19.375 −0.517 1.00 20.69 C ATOM 183 CD1 TYR 4810.643 18.539 −0.433 1.00 20.32 C ATOM 184 CE1 TYR 48 10.577 17.366−1.170 1.00 23.35 C ATOM 185 CD2 TYR 48 12.799 19.007 −1.362 1.00 20.03C ATOM 186 CE2 TYR 48 12.744 17.836 −2.107 1.00 22.54 C ATOM 187 CZ TYR48 11.631 17.021 −2.007 1.00 21.66 C ATOM 188 OH TYR 48 11.564 15.867−2.747 1.00 24.12 O ATOM 189 C TYR 48 11.761 23.093 0.444 1.00 18.48 CATOM 190 O TYR 48 12.732 23.838 0.420 1.00 18.96 O ATOM 191 N THR 4910.747 23.246 1.286 1.00 18.95 N ATOM 192 CA THR 49 10.792 24.247 2.3441.00 19.64 C ATOM 193 CB THR 49 9.398 24.779 2.729 1.00 20.36 C ATOM 194OG1 THR 49 8.595 23.704 3.229 1.00 18.68 O ATOM 195 CG2 THR 49 8.72025.421 1.531 1.00 21.70 C ATOM 196 C THR 49 11.332 23.403 3.503 1.0019.26 C ATOM 197 O THR 49 11.187 22.177 3.491 1.00 16.72 O ATOM 198 NMET 50 11.971 24.033 4.482 1.00 19.49 N ATOM 199 CA MET 50 12.496 23.2875.621 1.00 19.92 C ATOM 200 CB MET 50 13.164 24.227 6.623 1.00 19.27 CATOM 201 CG MET 50 13.752 23.549 7.857 1.00 21.93 C ATOM 202 SD MET 5015.038 22.346 7.482 1.00 22.54 S ATOM 203 CE MET 50 16.405 23.430 7.2201.00 21.56 C ATOM 204 C MET 50 11.366 22.505 6.292 1.00 20.51 C ATOM 205O MET 50 11.556 21.358 6.717 1.00 19.62 O ATOM 206 N LYS 51 10.18423.113 6.370 1.00 20.47 N ATOM 207 CA LYS 51 9.055 22.446 7.008 1.0021.99 C ATOM 208 CB LYS 51 7.860 23.388 7.165 1.00 26.64 C ATOM 209 CGLYS 51 6.659 22.735 7.834 1.00 33.29 C ATOM 210 CD LYS 51 5.483 23.6957.983 1.00 38.26 C ATOM 211 CE LYS 51 4.301 23.005 8.660 1.00 40.81 CATOM 212 NZ LYS 51 3.128 23.912 8.828 1.00 42.40 N ATOM 213 C LYS 518.650 21.172 6.273 1.00 19.90 C ATOM 214 O LYS 51 8.205 20.210 6.8981.00 19.38 O ATOM 215 N GLU 52 8.817 21.149 4.953 1.00 18.87 N ATOM 216CA GLU 52 8.464 19.944 4.205 1.00 17.93 C ATOM 217 CB GLU 52 8.38220.189 2.699 1.00 18.18 C ATOM 218 CG GLU 52 7.344 21.203 2.247 1.0023.24 C ATOM 219 CD GLU 52 7.396 21.313 0.732 1.00 25.60 C ATOM 220 OE1GLU 52 8.396 21.846 0.213 1.00 23.72 O ATOM 221 OE2 GLU 52 6.438 20.8740.057 1.00 26.89 O ATOM 222 C GLU 52 9.486 18.855 4.482 1.00 15.85 CATOM 223 O GLU 52 9.135 17.685 4.619 1.00 16.69 O ATOM 224 N VAL 5310.753 19.250 4.568 1.00 16.93 N ATOM 225 CA VAL 53 11.825 18.293 4.8251.00 17.04 C ATOM 226 CB VAL 53 13.203 18.995 4.855 1.00 16.03 C ATOM227 CG1 VAL 53 14.280 18.019 5.304 1.00 16.51 C ATOM 228 CG2 VAL 5313.534 19.547 3.467 1.00 17.18 C ATOM 229 C VAL 53 11.596 17.602 6.1661.00 15.46 C ATOM 230 O VAL 53 11.612 16.371 6.254 1.00 15.66 O ATOM 231N LEU 54 11.368 18.401 7.203 1.00 15.11 N ATOM 232 CA LEU 54 11.13617.868 8.539 1.00 18.02 C ATOM 233 CB LEU 54 11.038 19.004 9.564 1.0018.06 C ATOM 234 CG LEU 54 12.275 19.897 9.715 1.00 19.94 C ATOM 235 CD1LEU 54 12.017 21.075 10.651 1.00 21.06 C ATOM 236 CD2 LEU 54 13.48419.088 10.172 1.00 19.37 C ATOM 237 C LEU 54 9.887 16.989 8.551 1.0017.44 C ATOM 238 O LEU 54 9.890 15.901 9.132 1.00 17.96 O ATOM 239 N TYR55 8.824 17.456 7.900 1.00 19.29 N ATOM 240 CA TYR 55 7.582 16.686 7.8261.00 19.06 C ATOM 241 CB TYR 55 6.476 17.462 7.107 1.00 21.67 C ATOM 242CG TYR 55 5.208 16.659 6.914 1.00 24.34 C ATOM 243 CD1 TYR 55 4.33116.414 7.970 1.00 27.75 C ATOM 244 CE1 TYR 55 3.185 15.626 7.788 1.0028.11 C ATOM 245 CD2 TYR 55 4.910 16.100 5.671 1.00 26.71 C ATOM 246 CE2TYR 55 3.778 15.316 5.479 1.00 27.50 C ATOM 247 CZ TYR 55 2.920 15.0826.537 1.00 29.01 C ATOM 248 OH TYR 55 1.802 14.302 6.332 1.00 31.62 OATOM 249 C TYR 55 7.778 15.329 7.162 1.00 17.61 C ATOM 250 O TYR 557.450 14.295 7.742 1.00 18.75 O ATOM 251 N TYR 56 8.314 15.332 5.9431.00 17.90 N ATOM 252 CA TYR 56 8.537 14.079 5.228 1.00 15.80 C ATOM 253CB TYR 56 8.959 14.322 3.781 1.00 16.36 C ATOM 254 CG TYR 56 7.89714.995 2.944 1.00 19.38 C ATOM 255 CD1 TYR 56 6.589 14.508 2.934 1.0018.67 C ATOM 256 CE1 TYR 56 5.610 15.083 2.132 1.00 21.66 C ATOM 257 CD2TYR 56 8.201 16.083 2.125 1.00 19.49 C ATOM 258 CE2 TYR 56 7.225 16.6671.315 1.00 20.60 C ATOM 259 CZ TYR 56 5.935 16.160 1.325 1.00 22.09 CATOM 260 OH TYR 56 4.967 16.724 0.524 1.00 24.00 O ATOM 261 C TYR 569.463 13.067 5.893 1.00 16.42 C ATOM 262 O TYR 56 9.247 11.863 5.7591.00 16.20 O ATOM 263 N LEU 57 10.484 13.531 6.609 1.00 17.70 N ATOM 264CA LEU 57 11.362 12.579 7.283 1.00 18.25 C ATOM 265 CB LEU 57 12.62913.232 7.836 1.00 22.32 C ATOM 266 CG LEU 57 13.582 13.831 6.810 1.0026.55 C ATOM 267 CD1 LEU 57 14.757 14.543 7.473 1.00 27.16 C ATOM 268CD2 LEU 57 14.073 12.746 5.861 1.00 29.77 C ATOM 269 C LEU 57 10.58811.891 8.385 1.00 18.46 C ATOM 270 O LEU 57 10.782 10.704 8.641 1.0019.38 O ATOM 271 N GLY 58 9.693 12.639 9.023 1.00 16.71 N ATOM 272 CAGLY 58 8.883 12.060 10.077 1.00 18.09 C ATOM 273 C CLY 58 7.958 11.0309.458 1.00 18.69 C ATOM 274 O GLY 58 7.750 9.948 10.012 1.00 17.12 OATOM 275 N CLN 59 7.402 11.370 8.297 1.00 18.56 N ATOM 276 CA GLN 596.494 10.470 7.585 1.00 19.64 C ATOM 277 CB GLN 59 5.787 11.209 6.4481.00 21.81 C ATOM 278 CG GLN 59 4.918 12.363 6.913 1.00 25.45 C ATOM 279CD GLN 59 3.842 11.829 7.830 1.00 29.94 C ATOM 280 OE1 GLN 59 3.75312.211 9.000 1.00 32.36 O ATOM 281 NE2 GLN 59 3.015 10.934 7.304 1.0031.47 N ATOM 282 C GLN 59 7.212 9.228 7.062 1.00 18.28 C ATOM 283 O GLN59 6.624 8.150 6.977 1.00 18.50 O ATOM 284 N TYR 60 8.488 9.391 6.7251.00 17.72 N ATOM 285 CA TYR 60 9.322 8.294 6.237 1.00 19.36 C ATOM 286CB TYR 60 10.699 8.816 5.829 1.00 19.10 C ATOM 287 CG TYR 60 11.6757.746 5.386 1.00 21.10 C ATOM 288 CD1 TYR 60 11.634 7.221 4.095 1.0022.00 C ATOM 289 CE1 TYR 60 12.526 6.224 3.691 1.00 20.83 C ATOM 290 CD2TYR 60 12.635 7.247 6.269 1.00 21.10 C ATOM 291 CE2 TYR 60 13.532 6.2455.876 1.00 20.35 C ATOM 292 CZ TYR 60 13.469 5.740 4.585 1.00 20.69 CATOM 293 OH TYR 60 14.329 4.746 4.187 1.00 20.09 O ATOM 294 C TYR 609.474 7.246 7.335 1.00 19.51 C ATOM 295 O TYR 60 9.283 6.046 7.113 1.0018.29 O ATOM 296 N ILE 61 9.812 7.722 8.527 1.00 17.99 N ATOM 297 CA ILE61 10.012 6.851 9.675 1.00 19.08 C ATOM 298 CB ILE 61 10.521 7.68710.878 1.00 20.08 C ATOM 299 CG2 ILE 61 10.452 6.869 12.164 1.00 20.84 CATOM 300 CG1 ILE 61 11.945 8.179 10.586 1.00 18.04 C ATOM 301 CD1 ILE 6112.547 9.033 11.670 1.00 20.08 C ATOM 302 C ILE 61 8.709 6.155 10.0491.00 20.09 C ATOM 303 O ILE 61 8.692 4.975 10.406 1.00 19.98 O ATOM 304N MET 62 7.606 6.879 9.933 1.00 20.53 N ATOM 305 CA MET 62 6.330 6.30310.301 1.00 25.34 C ATOM 306 CB MET 62 5.305 7.398 10.599 1.00 28.26 CATOM 307 CG MET 62 3.943 6.874 11.011 1.00 34.36 C ATOM 308 SD MET 622.800 8.211 11.360 1.00 41.93 S ATOM 309 CE MET 62 2.064 8.447 9.7271.00 41.20 C ATOM 310 C MET 62 5.800 5.313 9.277 1.00 25.27 C ATOM 311 OMET 62 5.281 4.257 9.648 1.00 24.70 O ATOM 312 N THR 63 5.951 5.6357.997 1.00 25.57 N ATOM 313 CA THR 63 5.441 4.755 6.954 1.00 28.86 CATOM 314 CB THR 63 5.393 5.479 5.599 1.00 29.41 C ATOM 315 OG1 THR 634.566 6.641 5.722 1.00 33.31 O ATOM 316 CG2 THR 63 4.809 4.574 4.5261.00 28.44 C ATOM 317 C THR 63 6.247 3.473 6.810 1.00 27.27 C ATOM 318 OTHR 63 5.711 2.449 6.383 1.00 27.48 O ATOM 319 N LYS 64 7.526 3.5107.168 1.00 26.66 N ATOM 320 CA LYS 64 8.324 2.295 7.087 1.00 26.49 CATOM 321 CB LYS 64 9.730 2.517 6.523 1.00 26.55 C ATOM 322 CG LYS 649.740 3.088 5.117 1.00 29.11 C ATOM 323 CD LYS 64 11.150 3.247 4.5711.00 28.43 C ATOM 324 CE LYS 64 11.850 1.886 4.510 1.00 29.76 C ATOM 325NZ LYS 64 13.237 1.959 3.969 1.00 29.61 N ATOM 326 C LYS 64 8.375 1.6428.453 1.00 25.50 C ATOM 327 O LYS 64 9.026 0.617 8.639 1.00 26.13 O ATOM328 N ARG 65 7.678 2.255 9.403 1.00 24.76 N ATOM 329 CA ARG 65 7.6071.754 10.765 1.00 25.45 C ATOM 330 CB ARG 65 6.672 0.542 10.843 1.0031.11 C ATOM 331 CG ARG 65 5.246 0.879 10.397 1.00 35.38 C ATOM 332 CDARG 65 4.303 −0.309 10.470 1.00 41.28 C ATOM 333 NE ARG 65 2.954 0.05310.034 1.00 46.30 N ATOM 334 CZ ARG 65 1.897 −0.754 10.104 1.00 48.44 CATOM 335 NH1 ARG 65 2.023 −1.978 10.598 1.00 49.25 N ATOM 336 NH2 ARG 650.712 −0.338 9.674 1.00 48.82 N ATOM 337 C ARG 65 8.979 1.472 11.3781.00 25.07 C ATOM 338 O ARG 65 9.193 0.425 11.990 1.00 23.46 O ATOM 339N LEU 66 9.910 2.409 11.202 1.00 22.51 N ATOM 340 CA LEU 66 11.259 2.24611.745 1.00 21.93 C ATOM 341 CB LEU 66 12.251 3.184 11.063 1.00 21.83 CATOM 342 CG LEU 66 12.406 3.035 9.552 1.00 21.63 C ATOM 343 CD1 LEU 6613.391 4.062 9.015 1.00 20.95 C ATOM 344 CD2 LEU 66 12.838 1.634 9.1701.00 20.51 C ATOM 345 C LEU 66 11.322 2.436 13.259 1.00 22.70 C ATOM 346O LEU 66 12.325 2.115 13.893 1.00 22.69 O ATOM 347 N TYR 67 10.246 2.96213.833 1.00 23.65 N ATOM 348 CA TYR 67 10.174 3.180 15.273 1.00 23.29 CATOM 349 CB TYR 67 9.091 4.197 15.622 1.00 24.97 C ATOM 350 CG TYR 677.703 3.781 15.182 1.00 25.97 C ATOM 351 CD1 TYR 67 6.928 2.923 15.9661.00 27.64 C ATOM 352 CE1 TYR 67 5.656 2.522 15.554 1.00 27.94 C ATOM353 CD2 TYR 67 7.172 4.227 13.972 1.00 27.22 C ATOM 354 CE2 TYR 67 5.9053.830 13.549 1.00 28.73 C ATOM 355 CZ TYR 67 5.153 2.980 14.345 1.0029.04 C ATOM 356 OH TYR 67 3.899 2.594 13.930 1.00 29.83 O ATOM 357 CTYR 67 9.917 1.838 15.951 1.00 24.96 C ATOM 358 O TYR 67 9.215 0.99215.404 1.00 26.17 O ATOM 359 N ASP 68 10.500 1.629 17.124 1.00 25.95 NATOM 360 CA ASP 68 10.282 0.376 17.835 1.00 28.28 C ATOM 361 CB ASP 6811.205 0.225 19.038 1.00 31.87 C ATOM 362 CG ASP 68 10.969 −1.082 19.7811.00 35.20 C ATOM 363 OD1 ASP 68 10.683 −1.042 20.994 1.00 36.44 O ATOM364 OD2 ASP 68 11.061 −2.149 19.141 1.00 36.99 O ATOM 365 C ASP 68 8.8270.260 18.262 1.00 28.80 C ATOM 366 O ASP 68 8.286 1.157 18.907 1.0023.20 O ATOM 367 N GLU 69 8.200 −0.851 17.898 1.00 32.27 N ATOM 368 CAGLU 69 6.799 −1.073 18.224 1.00 36.70 C ATOM 369 CB GLU 69 6.336 −2.42817.684 1.00 40.26 C ATOM 370 CG GLU 69 4.873 −2.762 17.936 1.00 44.82 CATOM 371 CD GLU 69 3.981 −1.727 17.266 1.00 48.03 C ATOM 372 OE1 GLU 693.189 −2.115 16.380 1.00 50.17 O ATOM 373 OE2 GLU 69 4.064 −0.532 17.6211.00 50.09 O ATOM 374 C GLU 69 6.490 −0.956 19.719 1.00 36.29 C ATOM 375O GLU 69 5.376 −0.587 20.089 1.00 35.50 O ATOM 376 N LYS 70 7.471 −1.24520.574 1.00 36.45 N ATOM 377 CA LYS 70 7.246 −1.173 22.018 1.00 37.74 CATOM 378 CB LYS 70 7.591 −2.501 22.694 1.00 40.70 C ATOM 379 CG LYS 706.766 −3.677 22.186 1.00 43.93 C ATOM 380 CD LYS 70 7.137 −4.979 22.8891.00 46.97 C ATOM 381 CE LYS 70 6.298 −6.143 22.369 1.00 47.54 C ATOM382 NZ LYS 70 6.634 −7.434 23.042 1.00 48.98 N ATOM 383 C LYS 70 7.928−0.005 22.730 1.00 37.66 C ATOM 384 O LYS 70 7.557 0.348 23.852 1.0039.60 O ATOM 385 N GLN 71 8.918 0.595 22.080 1.00 33.78 N ATOM 386 CAGLN 71 9.615 1.749 22.643 1.00 31.65 C ATOM 387 CB GLN 71 11.073 1.42522.967 1.00 33.12 C ATOM 388 CG GLN 71 11.259 0.266 23.920 1.00 35.60 CATOM 389 CD GLN 71 12.739 0.054 24.162 1.00 36.23 C ATOM 390 OE1 GLN 7113.361 −0.811 23.548 1.00 35.70 O ATOM 391 NE2 GLN 71 13.305 0.82625.081 1.00 35.71 N ATOM 392 C GLN 71 9.530 2.706 21.466 1.00 29.31 CATOM 393 O GLN 71 10.488 2.864 20.713 1.00 23.70 O ATOM 394 N GLN 728.378 3.349 21.322 1.00 27.87 N ATOM 395 CA GLN 72 8.142 4.223 20.1851.00 29.09 C ATOM 396 CB GLN 72 6.650 4.530 20.041 1.00 32.14 C ATOM 397CG GLN 72 5.800 3.283 19.830 1.00 35.48 C ATOM 398 CD GLN 72 4.345 3.68019.685 1.00 37.97 C ATOM 399 OE1 GLN 72 3.689 3.324 18.707 1.00 40.02 OATOM 400 NE2 GLN 72 3.836 4.431 20.654 1.00 39.88 N ATOM 401 C GLN 728.975 5.480 20.002 1.00 27.04 C ATOM 402 O GLN 72 8.862 6.134 18.9681.00 28.04 O ATOM 403 N HIS 73 9.814 5.823 20.973 1.00 25.52 N ATOM 404CA HIS 73 10.638 7.017 20.817 1.00 26.50 C ATOM 405 CB HIS 73 10.8767.728 22.155 1.00 29.45 C ATOM 406 CG HIS 73 11.649 6.914 23.147 1.0033.08 C ATOM 407 CD2 HIS 73 11.240 6.122 24.167 1.00 34.44 C ATOM 408ND1 HIS 73 13.025 6.847 23.136 1.00 34.94 N ATOM 409 CE1 HIS 73 13.4326.048 24.109 1.00 35.16 C ATOM 410 NE2 HIS 73 12.369 5.596 24.748 1.0034.82 N ATOM 411 C HIS 73 11.968 6.628 20.170 1.00 25.41 C ATOM 412 OHIS 73 12.763 7.494 19.797 1.00 24.29 O ATOM 413 N ILE 74 12.200 5.32220.031 1.00 22.09 N ATOM 414 CA ILE 74 13.439 4.827 19.433 1.00 19.61 CATOM 415 CB ILE 74 13.967 3.575 20.159 1.00 19.95 C ATOM 416 CG2 ILE 7415.274 3.118 19.517 1.00 20.34 C ATOM 417 CG1 ILE 74 14.185 3.888 21.6401.00 15.63 C ATOM 418 CD1 ILE 74 14.696 2.712 22.442 1.00 20.76 C ATOM419 C ILE 74 13.275 4.472 17.966 1.00 20.12 C ATOM 420 O ILE 74 12.4403.642 17.593 1.00 19.78 O ATOM 421 N VAL 75 14.083 5.107 17.131 1.0018.20 N ATOM 422 CA VAL 75 14.034 4.856 15.703 1.00 17.89 C ATOM 423 CBVAL 75 14.193 6.171 14.901 1.00 17.74 C ATOM 424 CG1 VAL 75 14.278 5.86213.413 1.00 20.35 C ATOM 425 CG2 VAL 75 13.025 7.106 15.185 1.00 19.50 CATOM 426 C VAL 75 15.152 3.909 15.297 1.00 18.16 C ATOM 427 O VAL 7516.326 4.218 15.493 1.00 18.79 O ATOM 428 N HIS 76 14.788 2.754 14.7451.00 18.90 N ATOM 429 CA HIS 76 15.775 1.785 14.271 1.00 20.21 C ATOM430 CB HIS 76 15.334 0.332 14.487 1.00 19.35 C ATOM 431 CG HIS 76 15.229−0.064 15.924 1.00 18.87 C ATOM 432 CD2 HIS 76 14.203 0.011 16.804 1.0020.15 C ATOM 433 ND1 HIS 76 16.292 −0.602 16.618 1.00 17.09 N ATOM 434CE1 HIS 76 15.924 −0.841 17.864 1.00 18.81 C ATOM 435 NE2 HIS 76 14.661−0.478 18.003 1.00 18.94 N ATOM 436 C HIS 76 16.102 2.027 12.810 1.0021.94 C ATOM 437 O HIS 76 15.285 1.781 11.923 1.00 21.85 O ATOM 438 NCYS 77 17.314 2.496 12.567 1.00 19.97 N ATOM 439 CA CYS 77 17.743 2.78711.217 1.00 22.10 C ATOM 440 CB CYS 77 17.956 4.292 11.050 1.00 21.37 CATOM 441 SG CYS 77 19.086 4.999 12.249 1.00 23.82 S ATOM 442 C CYS 7718.889 1.937 10.685 1.00 22.14 C ATOM 443 O CYS 77 19.434 2.212 9.6191.00 21.97 O ATOM 444 N SER 78 19.251 0.899 11.434 1.00 22.19 N ATOM 445CA SER 78 20.304 −0.010 11.000 1.00 24.97 C ATOM 446 CB SER 78 20.689−1.008 12.098 1.00 24.27 C ATOM 447 OG SER 78 19.588 −1.819 12.475 1.0023.96 O ATOM 448 C SER 78 19.746 −0.727 9.772 1.00 27.08 C ATOM 449 OSER 78 18.578 −1.098 9.752 1.00 29.90 O ATOM 450 N ASN 79 20.562 −0.8968.739 1.00 31.08 N ATOM 451 CA ASN 79 20.102 −1.579 7.530 1.00 33.84 CATOM 452 CB ASN 79 19.475 −2.939 7.856 1.00 37.91 C ATOM 453 CG ASN 7920.430 −3.867 8.583 1.00 40.98 C ATOM 454 OD1 ASN 79 20.087 −4.436 9.6231.00 44.02 O ATOM 455 ND2 ASN 79 21.639 −4.014 8.050 1.00 41.58 N ATOM456 C ASN 79 19.130 −0.741 6.688 1.00 33.03 C ATOM 457 O ASN 79 18.500−1.260 5.765 1.00 31.20 O ATOM 458 N ASP 80 18.995 0.543 7.013 1.0028.81 N ATOM 459 CA ASP 80 18.098 1.425 6.263 1.00 24.65 C ATOM 460 CBASP 80 16.875 1.821 7.091 1.00 23.49 C ATOM 461 CG ASP 80 15.880 2.6626.307 1.00 24.25 C ATOM 462 OD1 ASP 80 14.807 2.115 5.975 1.00 23.20 OATOM 463 OD2 ASP 80 16.143 3.855 6.033 1.00 22.88 O ATOM 464 C ASP 8018.890 2.645 5.811 1.00 24.06 C ATOM 465 O ASP 80 19.814 3.078 6.5051.00 21.20 O ATOM 466 N LEU 81 18.546 3.188 4.647 1.00 21.17 N ATOM 467CA LEU 81 19.257 4.354 4.131 1.00 23.85 C ATOM 468 CB LEU 81 18.6384.843 2.819 1.00 27.45 C ATOM 469 CG LEU 81 19.280 6.057 2.138 1.0029.98 C ATOM 470 CD1 LEU 81 20.759 5.822 1.861 1.00 32.81 C ATOM 471 CD2LEU 81 18.548 6.421 0.845 1.00 32.11 C ATOM 472 C LEU 81 19.313 5.4865.155 1.00 21.14 C ATOM 473 O LEU 81 20.249 6.285 5.156 1.00 18.90 OATOM 474 N LEU 82 18.325 5.546 6.042 1.00 18.54 N ATOM 475 CA LEU 8218.318 6.598 7.052 1.00 15.44 C ATOM 476 CB LEU 82 17.019 6.590 7.8571.00 16.76 C ATOM 477 CG LEU 82 16.897 7.650 8.953 1.00 16.50 C ATOM 478CD1 LEU 82 17.090 9.052 8.382 1.00 17.63 C ATOM 479 CD2 LEU 82 15.5597.538 9.694 1.00 17.39 C ATOM 480 C LEU 82 19.516 6.405 7.969 1.00 15.53C ATOM 481 O LEU 82 20.070 7.373 8.495 1.00 15.91 O ATOM 482 N GLY 8319.916 5.145 8.139 1.00 14.77 N ATOM 483 CA GLY 83 21.058 4.824 8.9781.00 16.75 C ATOM 484 C GLY 83 22.341 5.353 8.365 1.00 19.93 C ATOM 485O GLY 83 23.236 5.824 9.075 1.00 19.30 O ATOM 486 N ASP 84 22.445 5.2787.042 1.00 20.23 N ATOM 487 CA ASP 84 23.640 5.790 6.376 1.00 19.77 CATOM 488 CB ASP 84 23.694 5.372 4.903 1.00 22.40 C ATOM 489 CG ASP 8423.784 3.865 4.712 1.00 24.25 C ATOM 490 OD1 ASP 84 24.006 3.137 5.7001.00 24.82 O ATOM 491 OD2 ASP 84 23.627 3.407 3.562 1.00 25.98 O ATOM492 C ASP 84 23.670 7.314 6.487 1.00 20.03 C ATOM 493 O ASP 84 24.7397.919 6.577 1.00 19.62 O ATOM 494 N LEU 85 22.488 7.925 6.511 1.00 16.11N ATOM 495 CA LEU 85 22.385 9.377 6.578 1.00 17.87 C ATOM 496 CB LEU 8520.972 9.827 6.194 1.00 19.94 C ATOM 497 CG LEU 85 20.673 11.320 6.0991.00 24.54 C ATOM 498 CD1 LEU 85 21.547 11.960 5.019 1.00 24.28 C ATOM499 CD2 LEU 85 19.205 11.575 5.770 1.00 24.55 C ATOM 500 C LEU 85 22.7799.897 7.965 1.00 18.33 C ATOM 501 O LEU 85 23.522 10.875 8.078 1.0018.30 O ATOM 502 N PHE 86 22.297 9.247 9.022 1.00 18.48 N ATOM 503 CAPHE 86 22.646 9.686 10.372 1.00 19.70 C ATOM 504 CB PHE 86 21.491 9.50611.370 1.00 22.89 C ATOM 505 CG PHE 86 20.283 10.375 11.092 1.00 25.20 CATOM 506 CD1 PHE 86 19.282 10.504 12.051 1.00 29.11 C ATOM 507 CD2 PHE86 20.175 11.114 9.915 1.00 26.71 C ATOM 508 CE1 PHE 86 18.195 11.35911.850 1.00 26.77 C ATOM 509 CE2 PHE 86 19.091 11.972 9.702 1.00 28.12 CATOM 510 CZ PHE 86 18.100 12.095 10.673 1.00 28.90 C ATOM 511 C PHE 8623.943 9.104 10.927 1.00 19.63 C ATOM 512 O PHE 86 24.464 9.594 11.9271.00 21.85 O ATOM 513 N GLY 87 24.468 8.074 10.272 1.00 18.21 N ATOM 514CA GLY 87 25.713 7.467 10.715 1.00 20.13 C ATOM 515 C GLY 87 25.6206.699 12.021 1.00 22.27 C ATOM 516 O GLY 87 26.612 6.552 12.737 1.0021.79 O ATOM 517 N VAL 88 24.425 6.210 12.332 1.00 21.59 N ATOM 518 CAVAL 88 24.186 5.448 13.555 1.00 22.13 C ATOM 519 CB VAL 88 23.716 6.35114.724 1.00 22.68 C ATOM 520 CG1 VAL 88 24.830 7.306 15.130 1.00 24.59 CATOM 521 CG2 VAL 88 22.463 7.117 14.324 1.00 23.26 C ATOM 522 C VAL 8823.105 4.408 13.305 1.00 21.88 C ATOM 523 O VAL 88 22.271 4.576 12.4171.00 21.47 O ATOM 524 N PRO 89 23.116 3.307 14.074 1.00 22.24 N ATOM 525CD PRO 89 23.982 2.960 15.221 1.00 23.24 C ATOM 526 CA PRO 89 22.1102.267 13.901 1.00 20.32 C ATOM 527 CB PRO 89 22.807 1.064 14.503 1.0023.05 C ATOM 528 CG PRO 89 23.286 1.669 15.787 1.00 22.08 C ATOM 529 CPRO 89 20.770 2.609 14.559 1.00 20.59 C ATOM 530 O PRO 89 19.775 1.91214.343 1.00 21.33 O ATOM 531 N SER 90 20.743 3.688 15.341 1.00 18.62 NATOM 532 CA SER 90 19.520 4.097 16.035 1.00 17.25 C ATOM 533 CB SER 9019.109 3.069 17.085 1.00 18.96 C ATOM 534 OG SER 90 20.118 2.972 18.0801.00 16.15 O ATOM 535 C SER 90 19.640 5.447 16.724 1.00 17.40 C ATOM 536O SER 90 20.742 5.937 16.990 1.00 17.90 O ATOM 537 N PHE 91 18.486 6.04017.013 1.00 15.60 N ATOM 538 CA PHE 91 18.422 7.308 17.718 1.00 16.44 CATOM 539 CB PHE 91 18.750 8.512 16.828 1.00 19.56 C ATOM 540 CG PHE 9117.793 8.708 15.682 1.00 19.09 C ATOM 541 CD1 PHE 91 17.923 7.972 14.5121.00 19.65 C ATOM 542 CD2 PHE 91 16.756 9.634 15.783 1.00 21.34 C ATOM543 CE1 PHE 91 17.033 8.153 13.450 1.00 20.79 C ATOM 544 CE2 PHE 9115.858 9.824 14.728 1.00 20.20 C ATOM 545 CZ PHE 91 15.999 9.083 13.5611.00 20.92 C ATOM 546 C PHE 91 17.043 7.491 18.334 1.00 18.30 C ATOM 547O PHE 91 16.058 6.939 17.846 1.00 17.76 O ATOM 548 N SER 92 16.992 8.24819.422 1.00 18.06 N ATOM 549 CA SER 92 15.737 8.575 20.083 1.00 20.21 CATOM 550 CB SER 92 15.913 8.741 21.587 1.00 20.33 C ATOM 551 OG SER 9214.676 9.099 22.181 1.00 22.41 O ATOM 552 C SER 92 15.224 9.875 19.4681.00 20.17 C ATOM 553 O SER 92 16.010 10.786 19.203 1.00 19.59 O ATOM554 N VAL 93 13.920 9.970 19.226 1.00 20.06 N ATOM 555 CA VAL 93 13.38111.198 18.647 1.00 22.08 C ATOM 556 CB VAL 93 11.867 11.074 18.371 1.0021.37 C ATOM 557 CG1 VAL 93 11.608 9.904 17.429 1.00 22.86 C ATOM 558CG2 VAL 93 11.114 10.891 19.681 1.00 22.21 C ATOM 559 C VAL 93 13.61412.384 19.583 1.00 22.18 C ATOM 560 O VAL 93 13.425 13.538 19.197 1.0020.43 O ATOM 561 N LYS 94 14.038 12.098 20.811 1.00 24.14 N ATOM 562 CALYS 94 14.298 13.153 21.784 1.00 25.77 C ATOM 563 CB LYS 94 14.14212.620 23.213 1.00 30.87 C ATOM 564 CG LYS 94 15.094 11.484 23.557 1.0036.14 C ATOM 565 CD LYS 94 14.888 10.965 24.976 1.00 40.51 C ATOM 566 CELYS 94 13.467 10.431 25.164 1.00 42.75 C ATOM 567 NZ LYS 94 13.224 9.90726.539 1.00 45.31 N ATOM 568 C LYS 94 15.677 13.781 21.558 1.00 24.88 CATOM 569 O LYS 94 15.992 14.825 22.129 1.00 24.06 O ATOM 570 N GLU 9516.488 13.144 20.715 1.00 23.25 N ATOM 571 CA GLU 95 17.823 13.65320.393 1.00 22.14 C ATOM 572 CB GLU 95 18.761 12.526 19.940 1.00 24.17 CATOM 573 CG GLU 95 19.031 11.470 21.011 1.00 28.59 C ATOM 574 CD GLU 9519.963 10.399 20.463 1.00 31.12 C ATOM 575 OE1 GLU 95 21.188 10.51120.674 1.00 33.36 O ATOM 576 OE2 GLU 95 19.473 9.430 19.847 1.00 32.84 OATOM 577 C GLU 95 17.733 14.757 19.346 1.00 20.96 C ATOM 578 O GLU 9518.275 14.639 18.245 1.00 18.40 O ATOM 579 N HIS 96 17.049 15.839 19.7021.00 20.19 N ATOM 580 CA HIS 96 16.860 16.952 18.782 1.00 20.60 C ATOM581 CB HIS 96 16.067 18.089 19.431 1.00 19.83 C ATOM 582 CG HIS 9614.680 17.702 19.844 1.00 21.58 C ATOM 583 CD2 HIS 96 13.987 16.55219.675 1.00 22.20 C ATOM 584 ND1 HIS 96 13.827 18.572 20.490 1.00 24.09N ATOM 585 CE1 HIS 96 12.667 17.975 20.700 1.00 23.77 C ATOM 586 NE2 HIS96 12.737 16.748 20.215 1.00 24.59 N ATOM 587 C HIS 96 18.105 17.50218.110 1.00 19.96 C ATOM 588 O HIS 96 18.112 17.693 16.897 1.00 18.54 OATOM 589 N ARG 97 19.163 17.750 18.873 1.00 19.16 N ATOM 590 CA ARG 9720.343 18.309 18.241 1.00 21.59 C ATOM 591 CB ARG 97 21.402 18.73319.254 1.00 23.22 C ATOM 592 CG ARG 97 22.605 19.332 18.560 1.00 26.59 CATOM 593 CD ARG 97 23.697 19.791 19.503 1.00 29.57 C ATOM 594 NE ARG 9724.804 20.353 18.733 1.00 30.13 N ATOM 595 CZ ARG 97 25.877 20.93019.263 1.00 31.77 C ATOM 596 NH1 ARG 97 25.998 21.028 20.581 1.00 30.89N ATOM 597 NH2 ARG 97 26.825 21.417 18.470 1.00 30.56 N ATOM 598 C ARG97 20.927 17.382 17.187 1.00 20.19 C ATOM 599 O ARG 97 21.341 17.83616.124 1.00 18.40 O ATOM 600 N LYS 98 20.945 16.083 17.472 1.00 20.40 NATOM 601 CA LYS 98 21.462 15.113 16.512 1.00 22.67 C ATOM 602 CB LYS 9821.499 13.710 17.125 1.00 25.02 C ATOM 603 CG LYS 98 21.910 12.59016.172 1.00 28.25 C ATOM 604 CD LYS 98 23.290 12.753 15.572 1.00 32.85 CATOM 605 CE LYS 98 23.591 11.577 14.640 1.00 35.25 C ATOM 606 NZ LYS 9824.936 11.653 13.997 1.00 38.28 N ATOM 607 C LYS 98 20.636 15.113 15.2321.00 21.36 C ATOM 608 O LYS 98 21.177 15.067 14.124 1.00 20.55 O ATOM609 N ILE 99 19.322 15.183 15.395 1.00 17.78 N ATOM 610 CA ILE 99 18.40915.168 14.261 1.00 16.72 C ATOM 611 CB ILE 99 16.962 15.036 14.757 1.0018.32 C ATOM 612 CG2 ILE 99 15.999 15.108 13.577 1.00 18.94 C ATOM 613CG1 ILE 99 16.813 13.713 15.522 1.00 18.48 C ATOM 614 CD1 ILE 99 15.43213.460 16.100 1.00 20.38 C ATOM 615 C ILE 99 18.556 16.406 13.379 1.0015.71 C ATOM 616 O ILE 99 18.745 16.289 12.166 1.00 16.59 O ATOM 617 NTYR 100 18.473 17.594 13.970 1.00 16.29 N ATOM 618 CA TYR 100 18.64918.801 13.168 1.00 15.73 C ATOM 619 CB TYR 100 18.384 20.081 13.959 1.0017.85 C ATOM 620 CG TYR 100 16.958 20.305 14.383 1.00 18.03 C ATOM 621CD1 TYR 100 15.989 20.628 13.433 1.00 18.59 C ATOM 622 CE1 TYR 10014.686 20.920 13.805 1.00 19.33 C ATOM 623 CD2 TYR 100 16.585 20.27215.724 1.00 19.08 C ATOM 624 CE2 TYR 100 15.277 20.564 16.108 1.00 20.31C ATOM 625 CZ TYR 100 14.337 20.889 15.142 1.00 20.85 C ATOM 626 OH TYR100 13.047 21.200 15.505 1.00 20.48 O ATOM 627 C TYR 100 20.028 18.89812.547 1.00 17.08 C ATOM 628 O TYR 100 20.177 19.362 11.417 1.00 17.10 OATOM 629 N THR 101 21.038 18.455 13.282 1.00 17.01 N ATOM 630 CA THR 10122.395 18.547 12.771 1.00 18.10 C ATOM 631 CB THR 101 23.421 18.06113.818 1.00 19.38 C ATOM 632 OG1 THR 101 23.409 18.950 14.944 1.00 19.40O ATOM 633 CG2 THR 101 24.827 18.023 13.219 1.00 21.23 C ATOM 634 C THR101 22.563 17.729 11.509 1.00 17.82 C ATOM 635 O THR 101 23.123 18.20710.523 1.00 18.96 O ATOM 636 N MET 102 22.055 16.503 11.515 1.00 14.87 NATOM 637 CA MET 102 22.241 15.673 10.345 1.00 18.86 C ATOM 638 CB MET102 22.051 14.200 10.687 1.00 22.58 C ATOM 639 CG MET 102 22.968 13.76911.831 1.00 25.73 C ATOM 640 SD MET 102 24.729 14.046 11.491 1.00 29.72S ATOM 641 CE MET 102 25.017 12.908 10.134 1.00 26.51 C ATOM 642 C MET102 21.366 16.115 9.181 1.00 17.36 C ATOM 643 O MET 102 21.716 15.9188.019 1.00 17.66 O ATOM 644 N ILE 103 20.242 16.746 9.488 1.00 17.10 NATOM 645 CA ILE 103 19.360 17.217 8.432 1.00 18.31 C ATOM 646 CB ILE 10317.971 17.598 9.004 1.00 18.43 C ATOM 647 CG2 ILE 103 17.137 18.3087.942 1.00 19.89 C ATOM 648 CG1 ILE 103 17.261 16.336 9.503 1.00 21.17 CATOM 649 CD1 ILE 103 15.895 16.591 10.129 1.00 21.70 C ATOM 650 C ILE103 19.997 18.442 7.778 1.00 18.58 C ATOM 651 O ILE 103 20.091 18.5216.552 1.00 18.12 O ATOM 652 N TYR 104 20.461 19.384 8.598 1.00 17.58 NATOM 653 CA TYR 104 21.063 20.600 8.065 1.00 19.92 C ATOM 654 CB TYR 10421.408 21.597 9.171 1.00 17.24 C ATOM 655 CG TYR 104 20.245 22.07310.015 1.00 19.35 C ATOM 656 CD1 TYR 104 18.920 21.924 9.590 1.00 17.10C ATOM 657 CE1 TYR 104 17.861 22.409 10.365 1.00 19.27 C ATOM 658 CD2TYR 104 20.479 22.714 11.227 1.00 17.95 C ATOM 659 CE2 TYR 104 19.43923.196 12.001 1.00 19.03 C ATOM 660 CZ TYR 104 18.137 23.044 11.571 1.0018.59 C ATOM 661 OH TYR 104 17.125 23.531 12.363 1.00 18.75 O ATOM 662 CTYR 104 22.308 20.332 7.224 1.00 18.77 C ATOM 663 O TYR 104 22.54421.020 6.234 1.00 17.01 O ATOM 664 N ARG 105 23.095 19.332 7.608 1.0019.90 N ATOM 665 CA ARG 105 24.303 19.019 6.850 1.00 22.21 C ATOM 666 CBARG 105 25.252 18.112 7.634 1.00 23.00 C ATOM 667 CG ARG 105 25.78618.721 8.916 1.00 25.04 C ATOM 668 CD ARG 105 26.724 17.753 9.616 1.0027.11 C ATOM 669 NE ARG 105 27.269 18.306 10.853 1.00 27.31 N ATOM 670CZ ARG 105 27.986 17.604 11.724 1.00 28.44 C ATOM 671 NH1 ARG 105 28.24116.324 11.488 1.00 27.73 N ATOM 672 NH2 ARG 105 28.442 18.175 12.8311.00 28.80 N ATOM 673 C ARG 105 24.007 18.433 5.479 1.00 22.30 C ATOM674 O ARG 105 24.893 18.333 4.632 1.00 24.22 O ATOM 675 N ASN 106 22.75218.054 5.267 1.00 20.53 N ATOM 676 CA ASN 106 22.317 17.497 3.993 1.0021.19 C ATOM 677 CB ASN 106 21.721 16.097 4.143 1.00 19.77 C ATOM 678 CGASN 106 22.744 15.084 4.612 1.00 22.79 C ATOM 679 OD1 ASN 106 23.42314.470 3.788 1.00 23.07 O ATOM 680 ND2 ASN 106 22.843 14.879 5.924 1.0019.66 N ATOM 681 C ASN 106 21.414 18.414 3.191 1.00 20.46 C ATOM 682 OASN 106 20.657 17.967 2.324 1.00 19.35 O ATOM 683 N LEU 107 21.51019.703 3.497 1.00 20.37 N ATOM 684 CA LEU 107 20.722 20.740 2.845 1.0022.38 C ATOM 685 CB LEU 107 19.663 21.303 3.791 1.00 21.77 C ATOM 686 CGLEU 107 18.618 20.323 4.316 1.00 23.03 C ATOM 687 CD1 LEU 107 17.70620.996 5.334 1.00 21.74 C ATOM 688 CD2 LEU 107 17.816 19.716 3.169 1.0022.14 C ATOM 689 C LEU 107 21.586 21.872 2.306 1.00 23.14 C ATOM 690 OLEU 107 22.618 22.205 2.884 1.00 22.39 O ATOM 691 N VAL 108 21.15822.445 1.189 1.00 23.48 N ATOM 692 CA VAL 108 21.846 23.569 0.566 1.0026.51 C ATOM 693 CB VAL 108 22.484 23.158 −0.776 1.00 28.25 C ATOM 694CG1 VAL 108 23.128 24.364 −1.432 1.00 31.06 C ATOM 695 CG2 VAL 10823.511 22.064 −0.543 1.00 30.49 C ATOM 696 C VAL 108 20.795 24.647 0.3041.00 25.74 C ATOM 697 O VAL 108 19.766 24.363 −0.301 1.00 24.47 O ATOM698 N VAL 109 21.042 25.871 0.767 1.00 25.08 N ATOM 699 CA VAL 10920.096 26.958 0.548 1.00 28.59 C ATOM 700 CB VAL 109 20.491 28.224 1.3251.00 30.38 C ATOM 701 CG1 VAL 109 19.457 29.320 1.088 1.00 30.40 C ATOM702 CG2 VAL 109 20.600 27.907 2.809 1.00 30.06 C ATOM 703 C VAL 10920.007 27.285 −0.942 1.00 31.09 C ATOM 704 O VAL 109 21.017 27.538−1.600 1.00 30.68 O ATOM 705 N VAL 110 18.784 27.273 −1.457 1.00 34.26 NATOM 706 CA VAL 110 18.497 27.512 −2.869 1.00 40.68 C ATOM 707 CB VAL110 17.092 26.951 −3.231 1.00 41.37 C ATOM 708 CG1 VAL 110 16.776 27.222−4.691 1.00 42.09 C ATOM 709 CG2 VAL 110 17.046 25.456 −2.948 1.00 41.30C ATOM 710 C VAL 110 18.586 28.950 −3.376 1.00 45.42 C ATOM 711 O VAL110 18.320 29.904 −2.643 1.00 45.46 O ATOM 712 N ASN 111 18.980 29.063−4.644 1.00 50.62 N ATOM 713 CA ASN 111 19.090 30.319 −5.380 1.00 54.41C ATOM 714 CB ASN 111 18.454 30.130 −6.764 1.00 57.01 C ATOM 715 CG ASN111 18.469 31.386 −7.600 1.00 59.53 C ATOM 716 OD1 ASN 111 17.413 31.886−7.991 1.00 60.59 O ATOM 717 ND2 ASN 111 19.660 31.900 −7.891 1.00 60.11N ATOM 718 C ASN 111 18.521 31.547 −4.662 1.00 54.78 C ATOM 719 O ASN111 17.400 31.982 −4.938 1.00 56.01 O ATOM 720 C1 SCH 996 −1.000 8.67415.423 1.00 58.94 ATOM 721 C2 SCH 996 0.074 7.820 16.068 1.00 58.85 ATOM722 O1 SCH 996 −0.100 7.261 17.153 1.00 59.47 ATOM 723 N1 SCH 996 1.2027.745 15.337 1.00 57.09 ATOM 724 C3 SCH 996 2.471 7.578 16.042 1.0055.53 ATOM 725 C4 SCH 996 3.102 8.922 16.429 1.00 58.30 ATOM 726 O2 SCH996 3.020 9.892 15.673 1.00 59.95 ATOM 727 N2 SCH 996 3.724 8.908 17.6311.00 61.62 ATOM 728 C5 SCH 996 4.020 10.154 18.329 1.00 63.29 ATOM 729C6 SCH 996 2.706 10.796 18.790 1.00 65.39 ATOM 730 O3 SCH 996 2.51612.011 18.716 1.00 65.59 ATOM 731 N3 SCH 996 1.822 9.901 19.269 1.0067.92 ATOM 732 C7 SCH 996 0.554 10.407 19.793 1.00 70.11 ATOM 733 C8 SCH996 0.346 9.699 20.930 1.00 70.97 ATOM 734 O4 SCH 996 0.223 10.15322.077 1.00 71.51 ATOM 735 O5 SCH 996 0.325 8.321 20.830 1.00 71.56 ATOM736 C9 SCH 996 3.406 6.688 15.192 1.00 49.97 ATOM 737 C10 SCH 996 −0.50410.428 18.687 1.00 70.56 ATOM 738 C11 SCH 996 −0.315 11.537 17.664 1.0071.51 ATOM 739 C12 SCH 996 −1.305 12.653 17.941 1.00 72.44 ATOM 740 O6SCH 996 −0.991 13.651 18.515 1.00 72.59 ATOM 741 O7 SCH 996 −2.55612.416 17.482 1.00 72.61 ATOM 742 C13 SCH 996 4.852 6.796 15.638 1.0044.76 ATOM 743 C14 SCH 996 5.890 7.608 15.051 1.00 41.72 ATOM 744 C15SCH 996 7.096 7.390 15.825 1.00 40.91 ATOM 745 N4 SCH 996 6.776 6.47216.833 1.00 40.79 ATOM 746 C16 SCH 996 5.458 6.131 16.715 1.00 43.06ATOM 747 C17 SCH 996 5.912 8.510 13.946 1.00 40.02 ATOM 748 C18 SCH 9967.115 9.193 13.606 1.00 37.25 ATOM 749 C19 SCH 996 8.298 8.983 14.3701.00 36.32 ATOM 750 C20 SCH 996 8.295 8.082 15.474 1.00 37.47 ATOM 751CL1 SCH 996 9.737 9.840 13.965 1.00 29.32 ATOM 752 C21 SCH 996 4.74711.137 17.396 1.00 62.87 ATOM 753 C22 SCH 996 4.877 9.854 19.566 1.0062.94 ATOM 754 C23 SCH 996 6.279 9.364 19.188 1.00 62.44 ATOM 755 C24SCH 996 6.166 10.673 17.043 1.00 62.33 ATOM 756 C25 SCH 996 7.006 10.35618.280 1.00 62.08 ATOM 757 C1 SCH 998 7.020 14.306 19.529 1.00 32.04ATOM 758 C2 SCH 998 8.132 14.220 18.499 1.00 30.99 ATOM 759 O1 SCH 9989.294 13.953 18.813 1.00 31.33 ATOM 760 N1 SCH 998 7.711 14.463 17.2431.00 28.90 ATOM 761 C3 SCH 998 8.715 14.626 16.190 1.00 28.20 ATOM 762C4 SCH 998 9.050 16.095 15.923 1.00 28.60 ATOM 763 O2 SCH 998 8.18416.880 15.527 1.00 28.06 ATOM 764 N2 SCH 998 10.342 16.402 16.159 1.0026.21 ATOM 765 C5 SCH 998 10.807 17.775 16.036 1.00 28.33 ATOM 766 C6SCH 998 9.978 18.673 16.963 1.00 33.18 ATOM 767 O3 SCH 998 9.634 19.80816.630 1.00 35.64 ATOM 768 N3 SCH 998 9.686 18.088 18.140 1.00 37.61ATOM 769 C7 SCH 998 8.697 18.731 19.006 1.00 42.54 ATOM 770 C8 SCH 9989.245 18.545 20.404 1.00 42.96 ATOM 771 O4 SCH 998 9.535 19.446 21.0491.00 44.48 ATOM 772 O5 SCH 998 9.382 17.262 20.693 1.00 43.12 ATOM 773C9 SCH 998 8.244 13.892 14.923 1.00 27.77 ATOM 774 C10 SCH 998 7.29018.240 18.661 1.00 44.92 ATOM 775 C11 SCH 998 6.730 18.812 17.368 1.0048.30 ATOM 776 C12 SCH 998 5.583 19.752 17.684 1.00 49.87 ATOM 777 O6SCH 998 5.601 20.902 17.365 1.00 51.11 ATOM 778 O7 SCH 998 4.554 19.17518.348 1.00 50.51 ATOM 779 C13 SCH 998 9.158 14.149 13.740 1.00 25.37ATOM 780 C14 SCH 998 10.480 13.618 13.499 1.00 25.02 ATOM 781 C15 SCH998 10.902 14.099 12.199 1.00 25.73 ATOM 782 N4 SCH 998 9.868 14.89511.699 1.00 25.53 ATOM 783 C16 SCH 998 8.852 14.918 12.609 1.00 25.55ATOM 784 C17 SCH 998 11.350 12.777 14.254 1.00 25.02 ATOM 785 C18 SCH998 12.617 12.399 13.724 1.00 25.30 ATOM 786 C19 SCH 998 13.020 12.85812.437 1.00 26.55 ATOM 787 C20 SCH 998 12.171 13.711 11.677 1.00 24.81ATOM 788 CL1 SCH 998 14.529 12.355 11.778 1.00 26.71 ATOM 789 C21 SCH998 10.639 18.264 14.593 1.00 26.42 ATOM 790 C22 SCH 998 12.279 17.85216.457 1.00 26.82 ATOM 791 C23 SCH 998 13.181 17.061 15.496 1.00 25.79ATOM 792 C24 SCH 998 11.554 17.510 13.617 1.00 24.84 ATOM 793 C25 SCH998 13.025 17.532 14.048 1.00 24.21 ATOM 794 C1 SCH 999 14.304 27.53313.501 1.00 20.77 ATOM 795 C2 SCH 999 14.911 27.965 12.179 1.00 20.52ATOM 796 O1 SCH 999 14.277 28.618 11.350 1.00 21.90 ATOM 797 N1 SCH 99916.183 27.557 12.024 1.00 19.32 ATOM 798 C3 SCH 999 16.883 27.892 10.7871.00 20.88 ATOM 799 C4 SCH 999 16.080 27.517 9.526 1.00 22.08 ATOM 800O2 SCH 999 15.657 26.372 9.370 1.00 21.93 ATOM 801 N2 SCH 999 15.89428.551 8.669 1.00 22.51 ATOM 802 C5 SCH 999 15.213 28.377 7.382 1.0024.88 ATOM 803 C6 SCH 999 13.709 28.188 7.602 1.00 26.16 ATOM 804 O3 SCH999 12.953 27.935 6.665 1.00 27.24 ATOM 805 N3 SCH 999 13.318 28.3318.879 1.00 27.90 ATOM 806 C7 SCH 999 11.886 28.201 9.143 1.00 31.35 ATOM807 C8 SCH 999 11.241 29.583 9.052 1.00 34.43 ATOM 808 O4 SCH 999 10.00629.558 9.037 1.00 35.86 ATOM 809 O5 SCH 999 11.994 30.531 8.919 1.0036.30 ATOM 810 C9 SCH 999 18.277 27.245 10.824 1.00 20.43 ATOM 811 C10SCH 999 11.653 27.295 10.349 1.00 31.35 ATOM 812 C11 SCH 999 12.06625.844 10.129 1.00 29.83 ATOM 813 C12 SCH 999 11.716 25.026 11.360 1.0032.05 ATOM 814 O6 SCH 999 12.531 24.381 11.942 1.00 31.48 ATOM 815 O7SCH 999 10.416 25.092 11.730 1.00 32.95 ATOM 816 C13 SCH 999 19.09327.591 9.595 1.00 21.99 ATOM 817 C14 SCH 999 19.603 26.675 8.606 1.0021.11 ATOM 818 C15 SCH 999 20.326 27.456 7.626 1.00 22.79 ATOM 819 N4SCH 999 20.243 28.793 8.029 1.00 23.21 ATOM 820 C16 SCH 999 19.51828.863 9.184 1.00 22.90 ATOM 821 C17 SCH 999 19.512 25.260 8.455 1.0021.66 ATOM 822 C18 SCH 999 20.126 24.621 7.341 1.00 23.05 ATOM 823 C19SCH 999 20.833 25.388 6.371 1.00 22.06 ATOM 824 C20 SCH 999 20.93726.803 6.511 1.00 22.85 ATOM 825 CL1 SCH 999 21.550 24.594 5.014 1.0026.59 ATOM 826 C21 SCH 999 15.747 27.140 6.639 1.00 23.66 ATOM 827 C22SCH 999 15.409 29.639 6.533 1.00 24.90 ATOM 828 C23 SCH 999 16.86729.820 6.129 1.00 24.75 ATOM 829 C24 SCH 999 17.176 27.321 6.103 1.0023.22 ATOM 830 C25 SCH 999 17.379 28.625 5.329 1.00 23.88 ATOM 831 OH2WAT 1001 3.395 9.112 −1.435 1.00 16.59 O ATOM 839 OH2 WAT 1002 24.77512.618 6.376 1.00 17.52 O ATOM 832 OH2 WAT 1003 18.514 −0.561 14.6221.00 18.25 O ATOM 835 OH2 WAT 1004 15.044 24.301 11.181 1.00 18.80 OATOM 856 OH2 WAT 1005 20.867 15.283 20.436 1.00 24.20 O ATOM 842 OH2 WAT1006 9.724 26.049 6.031 1.00 24.72 O ATOM 860 OH2 WAT 1007 6.074 13.93110.197 1.00 24.81 O ATOM 857 OH2 WAT 1008 21.757 8.195 18.608 1.00 25.80O ATOM 833 OH2 WAT 1009 12.107 26.746 4.500 1.00 25.85 O ATOM 843 OH2WAT 1010 16.620 1.807 2.942 1.00 26.33 O ATOM 870 OH2 WAT 1011 7.46620.307 9.569 1.00 26.55 O ATOM 848 OH2 WAT 1012 11.574 22.642 13.7791.00 26.80 O ATOM 834 OH2 WAT 1013 13.759 −1.012 20.605 1.00 28.49 OATOM 840 OH2 WAT 1014 25.743 20.101 15.817 1.00 28.57 O ATOM 855 OH2 WAT1015 0.544 11.546 5.002 1.00 29.35 O ATOM 844 OH2 WAT 1016 21.356 8.46022.469 1.00 30.69 O ATOM 836 OH2 WAT 1017 14.502 −0.707 6.212 1.00 31.40O ATOM 841 OH2 WAT 1018 11.775 14.532 17.517 1.00 31.42 O ATOM 851 OH2WAT 1019 22.475 1.722 7.609 1.00 31.99 O ATOM 859 OH2 WAT 1020 5.73218.551 −1.162 1.00 32.12 O ATOM 872 OH2 WAT 1021 23.725 20.929 22.6061.00 34.79 O ATOM 862 OH2 WAT 1022 23.860 26.433 1.644 1.00 34.96 O ATOM863 OH2 WAT 1023 5.394 16.356 11.571 1.00 35.71 O ATOM 861 OH2 WAT 102421.332 30.796 5.811 1.00 36.70 O ATOM 847 OH2 WAT 1025 9.148 −2.86315.900 1.00 37.64 O ATOM 858 OH2 WAT 1026 14.166 16.525 23.729 1.0037.84 O ATOM 866 OH2 WAT 1027 11.334 30.280 5.618 1.00 38.35 O ATOM 864OH2 WAT 1028 18.007 18.195 −4.362 1.00 40.37 O ATOM 871 OH2 WAT 102911.860 21.767 17.854 1.00 40.71 O ATOM 874 OH2 WAT 1030 8.276 27.4468.045 1.00 41.07 O ATOM 846 OH2 WAT 1031 7.632 18.152 11.360 1.00 42.63O ATOM 865 OH2 WAT 1032 25.898 10.512 16.626 1.00 43.37 O ATOM 854 OH2WAT 1033 8.159 −1.042 14.070 1.00 44.00 O ATOM 852 OH2 WAT 1034 28.46414.740 9.077 1.00 46.88 O ATOM 845 OH2 WAT 1035 2.078 6.181 4.658 1.0047.60 O ATOM 838 OH2 WAT 1036 26.484 15.271 4.182 1.00 47.99 O ATOM 867OH2 WAT 1037 23.657 28.439 −1.144 1.00 50.33 O ATOM 837 OH2 WAT 103813.538 21.361 20.180 1.00 54.13 O ATOM 869 OH2 WAT 1039 25.590 16.30916.444 1.00 54.19 O ATOM 850 OH2 WAT 1040 26.845 13.685 16.859 1.0055.86 O ATOM 873 OH2 WAT 1041 27.978 4.166 14.080 1.00 55.87 O ATOM 868OH2 WAT 1042 20.489 1.898 −2.662 1.00 56.16 O ATOM 853 OH2 WAT 104317.178 10.875 −6.741 1.00 59.20 O ATOM 849 OH2 WAT 1044 25.481 2.9068.796 1.00 63.46 O END

EXAMPLE 3 Preparation and Crystallization of Hdm2 Y76H-TripeptideComplex

Production and Crystallization of Modified Hdm2(Y76H):

The modified Hdm2(Y76H) protein was produced using the QuickChangesite-directed mutagenesis method as discussed in Example 1 except thatonly the primers for Y76H were used in mutagenesis. The p53 peptideanalog, Ac-^(6Cl)WAC_(3c)E, disclosed and defined above, was dissolvedin the same buffer and added to the Hdm2(Y76H) protein solution.

The single mutant HDM2 (17-125) Y76H-tripeptide complex was crystallizedusing a hanging-drop vapor diffusion method. The protein-peptidesolution (1 μl; 6-10 mg/ml) in buffer A was mixed with an equal volumeof precipitant [0.1 M Tris, pH 8-9, 35% PEG 4000, and 0.0 to 0.2 Mmagnesium chloride], placed on the underside of a siliconized glasscoverslip and sealed in close proximity to 1 ml of the precipitantsolution. Vapor diffusion crystallization experiments were conductedusing the hanging drop method. Specifically, crystals were grown from adroplet containing a mixture of 0.5-2.0 μl of protein and 0.5-1.0 μl ofthe precipitant solution. Crystallization plates were incubated at 4°C.; rectangular rod crystals (0.1×0.1×0.3 mm) grew over 2-30 days.

Prior to data collection, crystals were washed with the reservoirsolution of the crystallization setup and transferred into the samesolution with 10% glycerol added. The crystals were then flash-cooled ina nitrogen stream at 95 K. X-ray diffraction was collected using a Rigagenerator equipped with a Praxis 4++ detector. Data were integrated andscaled using the HKL package.

Data Collection Statistics:

Resolution 50.0-.20 Å No. of collected reflections 13234 No. of uniquereflections (F >= 0)  4341 R-sym  6.2% Percent of theoretical (I/s >= 1)84.8% Unit Cell a = 41.1 Å, b = 42.7 Å, c = 53.777 Å, α = β = γ = 90°Space Group P2₁2₁2₁ Asymmetric unit 1 molecule

The crystal structure was solved using molecular replacement using thesearch models 1YCQ and 1YCR from the PDB. Refinement was done using theprogram CNX.

Theoretical number of reflections 5854 Resolution Limits 50.0-2.1 ÅNumber of unobserved reflections 1529 (26.1%) Number of reflections inworking set 4325 (73.9%) Number of reflections in test set  197 (3.4%)Number of protein residues  87 Number of solvent atoms   0 R-factor  0.45 R-free   0.51 RMSD bond length     0.014 Å RMSD bond angles 1.97°

The structural coordinates for the above-described Hdm2 crystal are setforth below in Table 4.

TABLE 4 1 2 3 4 5 6 7 8 9 10 11 ATOM 1 CB GLU 25 7.387 −0.822 5.902 1.0031.40 C ATOM 2 CG GLU 25 7.019 −0.298 4.509 1.00 35.01 C ATOM 3 CD GLU25 7.873 −0.972 3.442 1.00 37.01 C ATOM 4 OE1 GLU 25 7.321 −1.714 2.6001.00 35.86 O ATOM 5 OE2 GLU 25 9.101 −0.744 3.437 1.00 38.68 O ATOM 6 CGLU 25 5.081 −0.176 6.662 1.00 30.27 C ATOM 7 O GLU 25 4.374 −1.1616.879 1.00 31.21 O ATOM 8 N GLU 25 6.922 −0.708 8.375 1.00 29.47 N ATOM9 CA GLU 25 6.567 −0.166 7.021 1.00 31.52 C ATOM 10 N THR 26 4.610 0.9386.116 1.00 28.18 N ATOM 11 CA THR 26 3.217 1.047 5.711 1.00 24.93 C ATOM12 CB THR 26 2.441 2.031 6.611 1.00 24.28 C ATOM 13 OG1 THR 26 2.6541.706 7.990 1.00 23.56 O ATOM 14 CG2 THR 26 0.955 1.959 6.304 1.00 22.48C ATOM 15 C THR 26 3.096 1.533 4.264 1.00 24.10 C ATOM 16 O THR 26 3.6332.581 3.908 1.00 22.83 O ATOM 17 N LEU 27 2.405 0.760 3.431 1.00 19.92 NATOM 18 CA LEU 27 2.177 1.160 2.047 1.00 20.55 C ATOM 19 CB LEU 27 2.052−0.044 1.107 1.00 22.12 C ATOM 20 CG LEU 27 3.257 −0.991 1.005 1.0024.55 C ATOM 21 CD1 LEU 27 2.945 −2.199 0.113 1.00 22.27 C ATOM 22 CD2LEU 27 4.506 −0.259 0.513 1.00 22.59 C ATOM 23 C LEU 27 0.899 1.9952.057 1.00 19.40 C ATOM 24 O LEU 27 −0.124 1.568 2.599 1.00 18.49 O ATOM25 N VAL 28 0.956 3.190 1.476 1.00 20.26 N ATOM 26 CA VAL 28 −0.2064.075 1.457 1.00 21.18 C ATOM 27 CB VAL 28 −0.090 5.200 2.523 1.00 19.01C ATOM 28 CG1 VAL 28 0.094 4.599 3.897 1.00 19.71 C ATOM 29 CG2 VAL 281.057 6.125 2.183 1.00 20.08 C ATOM 30 C VAL 28 −0.481 4.741 0.118 1.0021.98 C ATOM 31 O VAL 28 0.431 4.966 −0.679 1.00 23.19 O ATOM 32 N ARG29 −1.755 5.042 −0.120 1.00 23.38 N ATOM 33 CA ARG 29 −2.188 5.722−1.338 1.00 25.82 C ATOM 34 CB ARG 29 −3.239 4.904 −2.089 1.00 27.99 CATOM 35 CG ARG 29 −2.761 3.515 −2.489 1.00 30.54 C ATOM 36 CD ARG 29−3.838 2.738 −3.220 1.00 33.91 C ATOM 37 NE ARG 29 −3.398 1.395 −3.5901.00 37.13 N ATOM 38 CZ ARG 29 −2.478 1.134 −4.511 1.00 39.21 C ATOM 39NH1 ARG 29 −1.896 2.131 −5.168 1.00 41.39 N ATOM 40 NH2 ARG 29 −2.135−0.122 −4.771 1.00 39.25 N ATOM 41 C ARG 29 −2.743 7.082 −0.905 1.0026.04 C ATOM 42 O ARG 29 −3.723 7.163 −0.162 1.00 25.87 O ATOM 43 N PRO30 −2.100 8.167 −1.358 1.00 25.80 N ATOM 44 CD PRO 30 −0.679 8.055−1.738 1.00 24.67 C ATOM 45 CA PRO 30 −2.394 9.579 −1.111 1.00 22.90 CATOM 46 CB PRO 30 −1.203 10.259 −1.751 1.00 22.35 C ATOM 47 CG PRO 30−0.114 9.428 −1.255 1.00 26.65 C ATOM 48 C PRO 30 −3.699 10.094 −1.7231.00 22.50 C ATOM 49 O PRO 30 −4.062 9.701 −2.835 1.00 21.62 O ATOM 50 NLYS 31 −4.411 10.954 −1.001 1.00 22.08 N ATOM 51 CA LYS 31 −5.593 11.574−1.590 1.00 23.03 C ATOM 52 CB LYS 31 −6.451 12.381 −0.620 1.00 25.31 CATOM 53 CG LYS 31 −7.122 11.655 0.501 1.00 27.67 C ATOM 54 CD LYS 31−7.906 12.701 1.266 1.00 29.82 C ATOM 55 CE LYS 31 −8.654 12.122 2.4231.00 30.22 C ATOM 56 NZ LYS 31 −9.401 13.196 3.133 1.00 31.71 N ATOM 57C LYS 31 −5.065 12.527 −2.648 1.00 21.77 C ATOM 58 O LYS 31 −3.90612.951 −2.590 1.00 21.83 O ATOM 59 N PRO 32 −5.909 12.873 −3.629 1.0020.24 N ATOM 60 CD PRO 32 −7.253 12.301 −3.838 1.00 20.22 C ATOM 61 CAPRO 32 −5.608 13.770 −4.736 1.00 19.66 C ATOM 62 CB PRO 32 −6.996 14.120−5.227 1.00 21.15 C ATOM 63 CG PRO 32 −7.598 12.787 −5.277 1.00 21.31 CATOM 64 C PRO 32 −4.799 15.018 −4.348 1.00 18.96 C ATOM 65 O PRO 32−3.832 15.368 −5.022 1.00 17.76 O ATOM 66 N LEU 33 −5.190 15.682 −3.2631.00 20.65 N ATOM 67 CA LEU 33 −4.497 16.901 −2.853 1.00 22.91 C ATOM 68CB LEU 33 −5.412 17.805 −2.025 1.00 27.03 C ATOM 69 CG LEU 33 −6.69018.264 −2.733 1.00 30.44 C ATOM 70 CD1 LEU 33 −7.567 19.092 −1.796 1.0032.45 C ATOM 71 CD2 LEU 33 −6.385 19.036 −4.014 1.00 31.10 C ATOM 72 CLEU 33 −3.172 16.659 −2.141 1.00 22.13 C ATOM 73 O LEU 33 −2.277 17.507−2.169 1.00 20.13 O ATOM 74 N LEU 34 −3.041 15.500 −1.507 1.00 22.44 NATOM 75 CA LEU 34 −1.788 15.170 −0.845 1.00 22.32 C ATOM 76 CB LEU 34−1.967 14.032 0.158 1.00 22.46 C ATOM 77 CG LEU 34 −0.686 13.562 0.8511.00 25.03 C ATOM 78 CD1 LEU 34 0.092 14.709 1.488 1.00 22.68 C ATOM 79CD2 LEU 34 −0.984 12.449 1.846 1.00 25.15 C ATOM 80 C LEU 34 −0.83314.775 −1.959 1.00 20.98 C ATOM 81 O LEU 34 0.370 15.027 −1.892 1.0018.72 O ATOM 82 N LEU 35 −1.403 14.172 −2.996 1.00 22.16 N ATOM 83 CALEU 35 −0.644 13.746 −4.158 1.00 22.50 C ATOM 84 CB LEU 35 −1.499 12.888−5.086 1.00 21.79 C ATOM 85 CG LEU 35 −0.799 12.451 −6.371 1.00 21.31 CATOM 86 CD1 LEU 35 0.509 11.738 −6.069 1.00 22.29 C ATOM 87 CD2 LEU 35−1.709 11.605 −7.249 1.00 21.81 C ATOM 88 C LEU 35 −0.120 14.950 −4.9131.00 23.62 C ATOM 89 O LEU 35 0.965 14.909 −5.489 1.00 24.50 O ATOM 90 NLYS 36 −0.892 16.029 −4.887 1.00 26.45 N ATOM 91 CA LYS 36 −0.517 17.247−5.581 1.00 30.02 C ATOM 92 CB LYS 36 −1.739 18.156 −5.759 1.00 31.51 CATOM 93 CG LYS 36 −1.468 19.454 −6.494 1.00 34.42 C ATOM 94 CD LYS 36−2.742 20.280 −6.633 1.00 37.59 C ATOM 95 CE LYS 36 −2.483 21.584 −7.3811.00 40.02 C ATOM 96 NZ LYS 36 −3.723 22.409 −7.540 1.00 42.11 N ATOM 97C LYS 36 0.624 17.964 −4.864 1.00 30.99 C ATOM 98 O LYS 36 1.321 18.782−5.460 1.00 32.96 O ATOM 99 N LEU 37 0.828 17.645 −3.590 1.00 31.87 NATOM 100 CA LEU 37 1.909 18.274 −2.841 1.00 33.15 C ATOM 101 CB LEU 371.504 18.569 −1.395 1.00 34.78 C ATOM 102 CG LEU 37 0.316 19.498 −1.1441.00 37.22 C ATOM 103 CD1 LEU 37 0.039 19.625 0.347 1.00 36.79 C ATOM104 CD2 LEU 37 0.540 20.873 −1.760 1.00 38.28 C ATOM 105 C LEU 37 3.15317.395 −2.866 1.00 32.75 C ATOM 106 O LEU 37 4.277 17.889 −2.747 1.0031.66 O ATOM 107 N LEU 38 2.945 16.093 −3.040 1.00 30.45 N ATOM 108 CALEU 38 4.054 15.155 −3.080 1.00 28.31 C ATOM 109 CB LEU 38 3.578 13.726−2.805 1.00 27.70 C ATOM 110 CG LEU 38 2.878 13.360 −1.492 1.00 27.23 CATOM 111 CD1 LEU 38 2.383 11.925 −1.547 1.00 27.09 C ATOM 112 CD2 LEU 383.750 13.597 −0.262 1.00 24.95 C ATOM 113 C LEU 38 4.713 15.230 −4.4511.00 28.49 C ATOM 114 O LEU 38 5.888 14.897 −4.598 1.00 29.16 O ATOM 115N LYS 39 3.956 15.684 −5.449 1.00 27.07 N ATOM 116 CA LYS 39 4.48015.783 −6.806 1.00 28.73 C ATOM 117 CB LYS 39 3.373 15.754 −7.861 1.0033.27 C ATOM 118 CG LYS 39 2.504 14.521 −7.920 1.00 37.75 C ATOM 119 CDLYS 39 1.487 14.719 −9.035 1.00 41.51 C ATOM 120 CE LYS 39 0.550 13.539−9.187 1.00 45.31 C ATOM 121 NZ LYS 39 −0.430 13.767 −10.292 1.00 47.13N ATOM 122 C LYS 39 5.304 17.036 −7.034 1.00 25.77 C ATOM 123 O LYS 396.154 17.062 −7.916 1.00 27.98 O ATOM 124 N SER 40 5.064 18.071 −6.2401.00 24.34 N ATOM 125 CA SER 40 5.795 19.311 −6.427 1.00 23.75 C ATOM126 CB SER 40 5.033 20.501 −5.845 1.00 24.25 C ATOM 127 OG SER 40 4.84420.354 −4.451 1.00 24.47 O ATOM 128 C SER 40 7.180 19.229 −5.825 1.0023.75 C ATOM 129 O SER 40 8.029 20.066 −6.099 1.00 26.76 O ATOM 130 NVAL 41 7.414 18.203 −5.018 1.00 26.35 N ATOM 131 CA VAL 41 8.701 18.055−4.367 1.00 25.90 C ATOM 132 CB VAL 41 8.550 18.299 −2.846 1.00 26.80 CATOM 133 CG1 VAL 41 7.800 17.139 −2.204 1.00 25.77 C ATOM 134 CG2 VAL 419.905 18.491 −2.202 1.00 28.50 C ATOM 135 C VAL 41 9.308 16.674 −4.5971.00 26.12 C ATOM 136 O VAL 41 10.480 16.450 −4.300 1.00 28.42 O ATOM137 N GLY 42 8.523 15.751 −5.144 1.00 24.94 N ATOM 138 CA GLY 42 9.04914.416 −5.363 1.00 22.59 C ATOM 139 C GLY 42 8.438 13.615 −6.495 1.0022.27 C ATOM 140 O GLY 42 7.720 14.143 −7.347 1.00 19.19 O ATOM 141 NALA 43 8.740 12.321 −6.490 1.00 21.23 N ATOM 142 CA ALA 43 8.252 11.387−7.491 1.00 23.12 C ATOM 143 CB ALA 43 8.939 10.043 −7.326 1.00 22.78 CATOM 144 C ALA 43 6.740 11.202 −7.448 1.00 26.26 C ATOM 145 O ALA 436.137 11.093 −6.382 1.00 26.47 O ATOM 146 N GLN 44 6.144 11.182 −8.6321.00 29.51 N ATOM 147 CA GLN 44 4.711 10.994 −8.814 1.00 34.61 C ATOM148 CB GLN 44 4.254 11.798 −10.044 1.00 38.60 C ATOM 149 CG GLN 44 2.77211.752 −10.418 1.00 44.52 C ATOM 150 CD GLN 44 2.343 10.341 −10.743 1.0047.57 C ATOM 151 OE1 GLN 44 1.459 9.781 −10.093 1.00 50.77 O ATOM 152NE2 GLN 44 2.959 9.760 −11.768 1.00 49.38 N ATOM 153 C GLN 44 4.4529.486 −8.949 1.00 33.65 C ATOM 154 O GLN 44 4.970 8.844 −9.857 1.0034.09 O ATOM 155 N LYS 45 3.660 8.925 −8.040 1.00 33.88 N ATOM 156 CALYS 45 3.353 7.493 −8.071 1.00 34.47 C ATOM 157 CB LYS 45 4.586 6.652−7.744 1.00 31.48 C ATOM 158 CG LYS 45 5.205 6.911 −6.388 1.00 28.37 CATOM 159 CD LYS 45 6.411 6.017 −6.214 1.00 24.70 C ATOM 160 CE LYS 457.084 6.228 −4.883 1.00 24.73 C ATOM 161 NZ LYS 45 8.267 5.329 −4.7491.00 24.48 N ATOM 162 C LYS 45 2.151 7.096 −7.218 1.00 36.41 C ATOM 163O LYS 45 1.924 7.670 −6.157 1.00 36.79 O ATOM 164 N ASP 46 1.375 6.120−7.693 1.00 39.70 N ATOM 165 CA ASP 46 0.178 5.678 −6.974 1.00 41.06 CATOM 166 CB ASP 46 −0.641 4.672 −7.792 1.00 42.39 C ATOM 167 CG ASP 46−1.137 5.241 −9.105 1.00 43.83 C ATOM 168 OD1 ASP 46 −0.864 4.624−10.156 1.00 45.18 O ATOM 169 OD2 ASP 46 −1.799 6.301 −9.086 1.00 43.47O ATOM 170 C ASP 46 0.391 5.118 −5.573 1.00 40.30 C ATOM 171 O ASP 46−0.539 5.103 −4.769 1.00 40.91 O ATOM 172 N THR 47 1.605 4.671 −5.2681.00 38.48 N ATOM 173 CA THR 47 1.868 4.100 −3.953 1.00 35.27 C ATOM 174CB THR 47 1.860 2.564 −4.000 1.00 34.96 C ATOM 175 OG1 THR 47 0.5922.115 −4.482 1.00 35.07 O ATOM 176 CG2 THR 47 2.083 1.986 −2.614 1.0034.48 C ATOM 177 C THR 47 3.165 4.561 −3.312 1.00 33.33 C ATOM 178 O THR47 4.224 4.569 −3.942 1.00 34.15 O ATOM 179 N TYR 48 3.055 4.951 −2.0481.00 30.30 N ATOM 180 CA TYR 48 4.180 5.435 −1.256 1.00 27.10 C ATOM 181CB TYR 48 4.048 6.925 −0.949 1.00 24.18 C ATOM 182 CG TYR 48 3.995 7.837−2.150 1.00 21.88 C ATOM 183 CD1 TYR 48 5.152 8.418 −2.659 1.00 19.95 CATOM 184 CE1 TYR 48 5.096 9.298 −3.727 1.00 20.60 C ATOM 185 CD2 TYR 482.782 8.153 −2.747 1.00 19.91 C ATOM 186 CE2 TYR 48 2.716 9.027 −3.8121.00 20.90 C ATOM 187 CZ TYR 48 3.872 9.598 −4.298 1.00 20.62 C ATOM 188OH TYR 48 3.795 10.481 −5.347 1.00 22.83 O ATOM 189 C TYR 48 4.269 4.6850.061 1.00 26.72 C ATOM 190 O TYR 48 3.275 4.158 0.558 1.00 27.60 O ATOM191 N THR 49 5.470 4.620 0.616 1.00 27.57 N ATOM 192 CA THR 49 5.6474.001 1.916 1.00 27.42 C ATOM 193 CB THR 49 7.100 3.558 2.171 1.00 26.03C ATOM 194 OG1 THR 49 7.953 4.708 2.191 1.00 24.83 O ATOM 195 CG2 THR 497.562 2.595 1.092 1.00 26.14 C ATOM 196 C THR 49 5.317 5.167 2.836 1.0026.91 C ATOM 197 O THR 49 5.606 6.313 2.506 1.00 27.92 O ATOM 198 N MET50 4.692 4.891 3.969 1.00 28.56 N ATOM 199 CA MET 50 4.345 5.953 4.9051.00 30.04 C ATOM 200 CB MET 50 3.773 5.358 6.190 1.00 28.54 C ATOM 201CG MET 50 3.355 6.367 7.246 1.00 30.69 C ATOM 202 SD MET 50 2.058 7.4896.688 1.00 30.76 S ATOM 203 CE MET 50 2.888 9.022 6.804 1.00 31.08 CATOM 204 C MET 50 5.605 6.746 5.233 1.00 30.79 C ATOM 205 O MET 50 5.5467.896 5.664 1.00 31.44 O ATOM 206 N LYS 51 6.753 6.132 4.992 1.00 31.31N ATOM 207 CA LYS 51 8.008 6.764 5.343 1.00 31.72 C ATOM 208 CB LYS 518.996 5.654 5.711 1.00 34.50 C ATOM 209 CG LYS 51 10.325 6.042 6.2831.00 39.52 C ATOM 210 CD LYS 51 11.086 4.747 6.577 1.00 43.25 C ATOM 211CE LYS 51 12.449 4.991 7.201 1.00 46.68 C ATOM 212 NZ LYS 51 12.3185.719 8.503 1.00 47.10 N ATOM 213 C LYS 51 8.503 7.745 4.265 1.00 28.34C ATOM 214 O LYS 51 9.297 8.637 4.559 1.00 25.46 O ATOM 215 N GLU 528.011 7.600 3.032 1.00 24.36 N ATOM 216 CA GLU 52 8.388 8.523 1.956 1.0023.10 C ATOM 217 CB GLU 52 8.166 7.936 0.560 1.00 23.72 C ATOM 218 CGGLU 52 8.895 6.653 0.213 1.00 26.58 C ATOM 219 CD GLU 52 8.521 6.256−1.209 1.00 25.01 C ATOM 220 OE1 GLU 52 7.629 5.400 −1.371 1.00 24.55 OATOM 221 OE2 GLU 52 9.123 6.785 −2.164 1.00 25.12 O ATOM 222 C GLU 527.468 9.738 2.088 1.00 21.67 C ATOM 223 O GLU 52 7.843 10.860 1.740 1.0018.74 O ATOM 224 N VAL 53 6.263 9.495 2.599 1.00 18.59 N ATOM 225 CA VAL53 5.268 10.545 2.763 1.00 19.55 C ATOM 226 CB VAL 53 3.896 9.949 3.1431.00 19.33 C ATOM 227 CG1 VAL 53 2.896 11.062 3.434 1.00 19.57 C ATOM228 CG2 VAL 53 3.394 9.067 2.016 1.00 20.00 C ATOM 229 C VAL 53 5.70311.518 3.841 1.00 19.68 C ATOM 230 O VAL 53 5.559 12.727 3.684 1.0019.09 O ATOM 231 N LEU 54 6.253 10.983 4.926 1.00 21.56 N ATOM 232 CALEU 54 6.732 11.811 6.022 1.00 24.05 C ATOM 233 CB LEU 54 7.118 10.9497.227 1.00 25.58 C ATOM 234 CG LEU 54 5.962 10.130 7.805 1.00 27.53 CATOM 235 CD1 LEU 54 6.406 9.184 8.917 1.00 27.26 C ATOM 236 CD2 LEU 544.854 11.055 8.278 1.00 27.23 C ATOM 237 C LEU 54 7.917 12.628 5.5301.00 23.85 C ATOM 238 O LEU 54 8.066 13.800 5.876 1.00 24.62 O ATOM 239N PHE 55 8.747 12.002 4.702 1.00 22.58 N ATOM 240 CA PHE 55 9.908 12.6744.145 1.00 21.31 C ATOM 241 CB PHE 55 10.796 11.716 3.357 1.00 20.68 CATOM 242 CG PHE 55 11.981 12.387 2.725 1.00 20.30 C ATOM 243 CD1 PHE 5513.012 12.888 3.507 1.00 18.41 C ATOM 244 CD2 PHE 55 12.048 12.549 1.3461.00 19.84 C ATOM 245 CE1 PHE 55 14.085 13.538 2.928 1.00 18.68 C ATOM246 CE2 PHE 55 13.120 13.201 0.759 1.00 18.51 C ATOM 247 CZ PHE 5514.140 13.696 1.552 1.00 19.56 C ATOM 248 C PHE 55 9.512 13.850 3.2691.00 20.62 C ATOM 249 O PHE 55 9.963 14.971 3.493 1.00 23.48 O ATOM 250N TYR 56 8.670 13.599 2.270 1.00 19.21 N ATOM 251 CA TYR 56 8.243 14.6711.382 1.00 17.94 C ATOM 252 CB TYR 56 7.459 14.142 0.182 1.00 17.19 CATOM 253 CG TYR 56 8.259 13.265 −0.752 1.00 17.01 C ATOM 254 CD1 TYR 569.447 13.726 −1.310 1.00 16.35 C ATOM 255 CE1 TYR 56 10.130 12.987−2.251 1.00 15.96 C ATOM 256 CD2 TYR 56 7.781 12.024 −1.158 1.00 17.74 CATOM 257 CE2 TYR 56 8.457 11.273 −2.105 1.00 18.06 C ATOM 258 CZ TYR 569.627 11.763 −2.651 1.00 18.15 C ATOM 259 OH TYR 56 10.268 11.055 −3.6371.00 17.16 O ATOM 260 C TYR 56 7.506 15.830 2.034 1.00 18.40 C ATOM 261O TYR 56 7.708 16.974 1.632 1.00 20.22 O ATOM 262 N LEU 57 6.666 15.5593.034 1.00 18.61 N ATOM 263 CA LEU 57 5.964 16.652 3.699 1.00 19.85 CATOM 264 CB LEU 57 4.869 16.162 4.655 1.00 20.61 C ATOM 265 CG LEU 573.644 15.424 4.115 1.00 23.15 C ATOM 266 CD1 LEU 57 2.757 14.911 5.2431.00 23.67 C ATOM 267 CD2 LEU 57 2.843 16.320 3.162 1.00 21.07 C ATOM268 C LEU 57 6.942 17.560 4.425 1.00 19.93 C ATOM 269 O LEU 57 6.77818.777 4.429 1.00 20.07 O ATOM 270 N GLY 58 7.970 16.968 5.023 1.0018.96 N ATOM 271 CA GLY 58 8.959 17.764 5.724 1.00 19.61 C ATOM 272 CGLY 58 9.735 18.612 4.736 1.00 20.66 C ATOM 273 O GLY 58 9.973 19.8014.968 1.00 19.89 O ATOM 274 N GLN 59 10.132 18.003 3.622 1.00 21.02 NATOM 275 CA GLN 59 10.873 18.730 2.600 1.00 22.78 C ATOM 276 CB GLN 5911.519 17.776 1.599 1.00 22.59 C ATOM 277 CG GLN 59 12.505 16.839 2.2501.00 25.03 C ATOM 278 CD GLN 59 13.576 17.674 2.912 1.00 26.69 C ATOM279 OE1 GLN 59 13.877 17.502 4.090 1.00 28.42 O ATOM 280 NE2 GLN 5914.139 18.612 2.160 1.00 27.94 N ATOM 281 C GLN 59 9.980 19.745 1.9001.00 21.11 C ATOM 282 O GLN 59 10.458 20.730 1.342 1.00 20.60 O ATOM 283N TYR 60 8.675 19.504 1.943 1.00 20.96 N ATOM 284 CA TYR 60 7.725 20.4271.340 1.00 21.51 C ATOM 285 CB TYR 60 6.350 19.786 1.207 1.00 20.18 CATOM 286 CG TYR 60 5.290 20.727 0.684 1.00 19.26 C ATOM 287 CD1 TYR 605.127 20.946 −0.677 1.00 18.65 C ATOM 288 CE1 TYR 60 4.145 21.796 −1.1481.00 18.43 C ATOM 289 CD2 TYR 60 4.444 21.390 1.563 1.00 18.15 C ATOM290 CE2 TYR 60 3.465 22.238 1.106 1.00 19.04 C ATOM 291 CZ TYR 60 3.31422.437 −0.251 1.00 19.66 C ATOM 292 OH TYR 60 2.316 23.266 −0.705 1.0017.66 O ATOM 293 C TYR 60 7.631 21.661 2.221 1.00 21.23 C ATOM 294 O TYR60 7.592 22.793 1.737 1.00 21.02 O ATOM 295 N ILE 61 7.615 21.414 3.5251.00 20.45 N ATOM 296 CA ILE 61 7.514 22.463 4.518 1.00 19.88 C ATOM 297CB ILE 61 7.305 21.841 5.913 1.00 16.63 C ATOM 298 CG2 ILE 61 7.40522.907 6.990 1.00 15.17 C ATOM 299 CG1 ILE 61 5.961 21.112 5.956 1.0014.67 C ATOM 300 CD1 ILE 61 5.660 20.431 7.295 1.00 13.50 C ATOM 301 CILE 61 8.789 23.293 4.518 1.00 24.11 C ATOM 302 O ILE 61 8.779 24.4634.915 1.00 24.81 O ATOM 303 N MET 62 9.882 22.695 4.052 1.00 27.53 NATOM 304 CA MET 62 11.158 23.399 4.010 1.00 31.34 C ATOM 305 CB MET 6212.331 22.454 4.293 1.00 34.73 C ATOM 306 CG MET 62 12.295 21.791 5.6651.00 41.21 C ATOM 307 SD MET 62 13.705 20.685 5.949 1.00 45.94 S ATOM308 CE MET 62 15.034 21.883 6.155 1.00 46.15 C ATOM 309 C MET 62 11.39424.154 2.710 1.00 30.78 C ATOM 310 O MET 62 11.975 25.236 2.723 1.0031.93 O ATOM 311 N THR 63 10.929 23.607 1.591 1.00 30.29 N ATOM 312 CATHR 63 11.146 24.278 0.318 1.00 31.93 C ATOM 313 CB THR 63 10.913 23.331−0.879 1.00 32.45 C ATOM 314 OG1 THR 63 11.653 22.121 −0.691 1.00 34.57O ATOM 315 CG2 THR 63 11.383 23.992 −2.168 1.00 33.58 C ATOM 316 C THR63 10.222 25.476 0.164 1.00 30.66 C ATOM 317 O THR 63 10.550 26.430−0.535 1.00 32.12 O ATOM 318 N LYS 64 9.072 25.437 0.825 1.00 30.47 NATOM 319 CA LYS 64 8.144 26.554 0.750 1.00 30.88 C ATOM 320 CB LYS 646.715 26.113 0.433 1.00 31.95 C ATOM 321 CG LYS 64 6.592 25.412 −0.9031.00 33.47 C ATOM 322 CD LYS 64 5.163 25.021 −1.188 1.00 35.44 C ATOM323 CE LYS 64 4.269 26.251 −1.223 1.00 35.32 C ATOM 324 NZ LYS 64 2.85125.905 −1.507 1.00 36.49 N ATOM 325 C LYS 64 8.218 27.370 2.021 1.0030.38 C ATOM 326 O LYS 64 7.504 28.356 2.190 1.00 31.61 O ATOM 327 N ARG65 9.096 26.929 2.914 1.00 31.08 N ATOM 328 CA ARG 65 9.350 27.604 4.1761.00 32.41 C ATOM 329 CB ARG 65 10.296 28.776 3.921 1.00 33.62 C ATOM330 CG ARG 65 11.569 28.220 3.304 1.00 38.45 C ATOM 331 CD ARG 65 12.66029.194 2.949 1.00 39.91 C ATOM 332 NE ARG 65 13.766 28.425 2.375 1.0042.85 N ATOM 333 CZ ARG 65 14.793 28.944 1.713 1.00 43.34 C ATOM 334 NH1ARG 65 14.878 30.255 1.530 1.00 45.53 N ATOM 335 NH2 ARG 65 15.72928.146 1.221 1.00 43.64 N ATOM 336 C ARG 65 8.099 27.982 4.955 1.0030.80 C ATOM 337 O ARG 65 7.856 29.145 5.259 1.00 28.75 O ATOM 338 N LEU66 7.310 26.965 5.274 1.00 31.27 N ATOM 339 CA LEU 66 6.077 27.149 6.0201.00 31.53 C ATOM 340 CB LEU 66 5.082 26.044 5.687 1.00 30.56 C ATOM 341CG LEU 66 4.742 25.973 4.201 1.00 30.97 C ATOM 342 CD1 LEU 66 3.80024.809 3.914 1.00 31.85 C ATOM 343 CD2 LEU 66 4.156 27.290 3.700 1.0028.41 C ATOM 344 C LEU 66 6.391 27.171 7.507 1.00 32.00 C ATOM 345 O LEU66 5.505 27.320 8.342 1.00 36.99 O ATOM 346 N TYR 67 7.669 27.018 7.8221.00 32.16 N ATOM 347 CA TYR 67 8.147 27.010 9.196 1.00 29.34 C ATOM 348CB TYR 67 9.300 26.023 9.365 1.00 30.19 C ATOM 349 CG TYR 67 10.50526.362 8.517 1.00 30.32 C ATOM 350 CD1 TYR 67 11.362 27.393 8.877 1.0031.21 C ATOM 351 CE1 TYR 67 12.453 27.726 8.097 1.00 31.23 C ATOM 352CD2 TYR 67 10.775 25.668 7.348 1.00 31.49 C ATOM 353 CE2 TYR 67 11.86725.995 6.559 1.00 31.76 C ATOM 354 CZ TYR 67 12.702 27.025 6.939 1.0030.71 C ATOM 355 OH TYR 67 13.790 27.351 6.163 1.00 30.69 O ATOM 356 CTYR 67 8.584 28.410 9.599 1.00 26.76 C ATOM 357 O TYR 67 8.974 29.2128.755 1.00 27.18 O ATOM 358 N ASP 68 8.505 28.710 10.887 1.00 26.00 NATOM 359 CA ASP 68 8.931 30.012 11.376 1.00 24.98 C ATOM 360 CB ASP 688.536 30.213 12.832 1.00 24.44 C ATOM 361 CG ASP 68 8.916 31.581 13.3541.00 25.82 C ATOM 362 OD1 ASP 68 8.229 32.064 14.280 1.00 27.13 O ATOM363 OD2 ASP 68 9.876 32.189 12.829 1.00 23.05 O ATOM 364 C ASP 68 10.45630.065 11.239 1.00 26.08 C ATOM 365 O ASP 68 11.171 29.230 11.809 1.0020.81 O ATOM 366 N GLU 69 10.954 31.038 10.481 1.00 26.25 N ATOM 367 CAGLU 69 12.393 31.179 10.299 1.00 27.50 C ATOM 368 CB GLU 69 12.71132.341 9.355 1.00 31.03 C ATOM 369 CG GLU 69 14.200 32.606 9.160 1.0036.70 C ATOM 370 CD GLU 69 14.885 31.383 8.579 1.00 40.21 C ATOM 371 OE1GLU 69 14.189 30.407 8.224 1.00 42.79 O ATOM 372 OE2 GLU 69 16.13331.400 8.486 1.00 41.52 O ATOM 373 C GLU 69 13.155 31.330 11.617 1.0025.94 C ATOM 374 O GLU 69 14.320 30.956 11.705 1.00 25.86 O ATOM 375 NLYS 70 12.493 31.856 12.645 1.00 23.46 N ATOM 376 CA LYS 70 13.14132.048 13.937 1.00 23.45 C ATOM 377 CB LYS 70 12.882 33.449 14.490 1.0026.70 C ATOM 378 CG LYS 70 13.362 34.567 13.582 1.00 28.84 C ATOM 379 CDLYS 70 14.858 34.477 13.329 1.00 31.67 C ATOM 380 CE LYS 70 15.32735.607 12.415 1.00 33.30 C ATOM 381 NZ LYS 70 16.788 35.549 12.150 1.0034.03 N ATOM 382 C LYS 70 12.762 30.989 14.965 1.00 22.06 C ATOM 383 OLYS 70 13.147 31.077 16.123 1.00 21.58 O ATOM 384 N GLN 71 11.995 29.99314.541 1.00 21.15 N ATOM 385 CA GLN 71 11.588 28.913 15.436 1.00 20.20 CATOM 386 CB GLN 71 10.497 29.383 16.407 1.00 20.24 C ATOM 387 CG GLN 7110.110 28.357 17.460 1.00 20.50 C ATOM 388 CD GLN 71 9.029 28.928 18.3631.00 20.46 C ATOM 389 OE1 GLN 71 8.365 29.908 18.019 1.00 24.60 O ATOM390 NE2 GLN 71 8.865 28.330 19.535 1.00 18.17 N ATOM 391 C GLN 71 11.08827.853 14.461 1.00 20.18 C ATOM 392 O GLN 71 9.880 27.643 14.280 1.0020.12 O ATOM 393 N GLN 72 12.056 27.188 13.840 1.00 20.81 N ATOM 394 CAGLN 72 11.806 26.189 12.813 1.00 22.77 C ATOM 395 CB GLN 72 13.11925.855 12.110 1.00 23.53 C ATOM 396 CG GLN 72 13.723 27.086 11.456 1.0027.22 C ATOM 397 CD GLN 72 15.027 26.744 10.776 1.00 29.38 C ATOM 398OE1 GLN 72 15.497 25.604 10.824 1.00 30.31 O ATOM 399 NE2 GLN 72 15.62627.741 10.137 1.00 30.16 N ATOM 400 C GLN 72 11.028 24.940 13.195 1.0021.80 C ATOM 401 O GLN 72 10.603 24.178 12.328 1.00 20.25 O ATOM 402 NHIS 73 10.829 24.737 14.488 1.00 20.18 N ATOM 403 CA HIS 73 10.07623.585 14.949 1.00 21.21 C ATOM 404 CB HIS 73 10.627 23.080 16.287 1.0022.75 C ATOM 405 CG HIS 73 10.662 24.117 17.366 1.00 24.88 C ATOM 406CD2 HIS 73 11.670 24.911 17.801 1.00 25.16 C ATOM 407 ND1 HIS 73 9.56524.435 18.136 1.00 26.05 N ATOM 408 CE1 HIS 73 9.895 25.378 19.001 1.0027.37 C ATOM 409 NE2 HIS 73 11.167 25.684 18.818 1.00 27.33 N ATOM 410 CHIS 73 8.569 23.857 14.955 1.00 19.30 C ATOM 411 O HIS 73 7.762 23.01815.353 1.00 15.05 O ATOM 412 N ILE 74 8.202 25.049 14.495 1.00 20.68 NATOM 413 CA ILE 74 6.800 25.442 14.408 1.00 20.38 C ATOM 414 CB ILE 746.517 26.761 15.170 1.00 17.27 C ATOM 415 CG2 ILE 74 5.082 27.186 14.9301.00 16.94 C ATOM 416 CG1 ILE 74 6.788 26.585 16.673 1.00 12.85 C ATOM417 CD1 ILE 74 5.940 25.522 17.350 1.00 10.25 C ATOM 418 C ILE 74 6.43325.631 12.931 1.00 22.21 C ATOM 419 O ILE 74 7.068 26.401 12.207 1.0019.54 O ATOM 420 N VAL 75 5.406 24.914 12.489 1.00 22.66 N ATOM 421 CAVAL 75 4.968 24.994 11.106 1.00 20.85 C ATOM 422 CB VAL 75 4.656 23.59610.552 1.00 19.84 C ATOM 423 CG1 VAL 75 4.268 23.697 9.081 1.00 17.74 CATOM 424 CG2 VAL 75 5.870 22.678 10.745 1.00 19.97 C ATOM 425 C VAL 753.730 25.866 10.931 1.00 22.04 C ATOM 426 O VAL 75 2.682 25.605 11.5231.00 19.88 O ATOM 427 N HIS 76 3.860 26.907 10.117 1.00 19.99 N ATOM 428CA HIS 76 2.742 27.797 9.850 1.00 20.86 C ATOM 429 CB HIS 76 3.14029.249 10.074 1.00 20.76 C ATOM 430 CG HIS 76 3.595 29.532 11.471 1.0018.48 C ATOM 431 CD2 HIS 76 4.799 29.914 11.958 1.00 17.24 C ATOM 432ND1 HIS 76 2.764 29.401 12.561 1.00 18.78 N ATOM 433 CE1 HIS 76 3.43629.693 13.661 1.00 17.42 C ATOM 434 NE2 HIS 76 4.673 30.007 13.323 1.0017.51 N ATOM 435 C HIS 76 2.250 27.541 8.429 1.00 22.01 C ATOM 436 O HIS76 2.906 27.886 7.448 1.00 20.06 O ATOM 437 N CYS 77 1.076 26.934 8.3331.00 25.10 N ATOM 438 CA CYS 77 0.503 26.582 7.049 1.00 27.53 C ATOM 439CB CYS 77 0.499 25.055 6.882 1.00 26.53 C ATOM 440 SG CYS 77 −0.32124.144 8.202 1.00 29.08 S ATOM 441 C CYS 77 −0.805 27.255 6.632 1.0028.98 C ATOM 442 O CYS 77 −1.442 26.851 5.660 1.00 28.62 O ATOM 443 NSER 78 −1.206 28.283 7.377 1.00 31.46 N ATOM 444 CA SER 78 −2.404 29.0437.035 1.00 34.18 C ATOM 445 CB SER 78 −2.859 29.936 8.186 1.00 32.67 CATOM 446 OG SER 78 −1.858 30.878 8.515 1.00 32.54 O ATOM 447 C SER 78−1.935 29.883 5.851 1.00 37.37 C ATOM 448 O SER 78 −0.941 30.602 5.9661.00 40.89 O ATOM 449 N ASN 79 −2.637 29.782 4.725 1.00 38.16 N ATOM 450CA ASN 79 −2.281 30.491 3.490 1.00 38.25 C ATOM 451 CB ASN 79 −1.49031.788 3.729 1.00 41.43 C ATOM 452 CG ASN 79 −2.269 32.814 4.535 1.0044.32 C ATOM 453 OD1 ASN 79 −1.804 33.293 5.568 1.00 47.01 O ATOM 454ND2 ASN 79 −3.464 33.155 4.062 1.00 45.45 N ATOM 455 C ASN 79 −1.53529.579 2.522 1.00 36.47 C ATOM 456 O ASN 79 −1.050 30.026 1.481 1.0036.50 O ATOM 457 N ASP 80 −1.439 28.301 2.879 1.00 33.49 N ATOM 458 CAASP 80 −0.782 27.304 2.038 1.00 30.65 C ATOM 459 CB ASP 80 0.597 26.9082.567 1.00 28.08 C ATOM 460 CG ASP 80 1.300 25.894 1.666 1.00 27.51 CATOM 461 OD1 ASP 80 2.347 26.248 1.088 1.00 28.18 O ATOM 462 OD2 ASP 800.818 24.749 1.528 1.00 24.49 O ATOM 463 C ASP 80 −1.696 26.089 1.9261.00 30.18 C ATOM 464 O ASP 80 −2.399 25.750 2.876 1.00 32.29 O ATOM 465N LEU 81 −1.701 25.447 0.763 1.00 29.44 N ATOM 466 CA LEU 81 −2.54724.279 0.548 1.00 28.49 C ATOM 467 CB LEU 81 −2.242 23.632 −0.806 1.0030.75 C ATOM 468 CG LEU 81 −3.069 22.396 −1.179 1.00 32.89 C ATOM 469CD1 LEU 81 −4.564 22.720 −1.167 1.00 32.68 C ATOM 470 CD2 LEU 81 −2.65821.821 −2.530 1.00 33.32 C ATOM 471 C LEU 81 −2.428 23.240 1.667 1.0027.48 C ATOM 472 O LEU 81 −3.327 22.422 1.861 1.00 26.60 O ATOM 473 NLEU 82 −1.328 23.284 2.411 1.00 25.83 N ATOM 474 CA LEU 82 −1.121 22.3263.485 1.00 25.93 C ATOM 475 CB LEU 82 0.335 22.334 3.953 1.00 27.37 CATOM 476 CG LEU 82 0.714 21.341 5.051 1.00 26.40 C ATOM 477 CD1 LEU 820.391 19.918 4.617 1.00 28.04 C ATOM 478 CD2 LEU 82 2.183 21.459 5.4401.00 25.48 C ATOM 479 C LEU 82 −2.075 22.622 4.640 1.00 26.61 C ATOM 480O LEU 82 −2.394 21.736 5.434 1.00 24.04 O ATOM 481 N GLY 83 −2.53923.870 4.713 1.00 25.97 N ATOM 482 CA GLY 83 −3.476 24.255 5.751 1.0023.73 C ATOM 483 C GLY 83 −4.816 23.613 5.437 1.00 24.97 C ATOM 484 OGLY 83 −5.531 23.137 6.324 1.00 23.63 O ATOM 485 N ASP 84 −5.155 23.5924.154 1.00 26.10 N ATOM 486 CA ASP 84 −6.404 22.994 3.705 1.00 28.17 CATOM 487 CB ASP 84 −6.695 23.378 2.259 1.00 29.18 C ATOM 488 CG ASP 84−6.829 24.868 2.065 1.00 31.27 C ATOM 489 OD1 ASP 84 −6.896 25.604 3.0731.00 30.35 O ATOM 490 OD2 ASP 84 −6.856 25.298 0.891 1.00 33.57 O ATOM491 C ASP 84 −6.345 21.477 3.817 1.00 28.68 C ATOM 492 O ASP 84 −7.36920.818 3.992 1.00 29.33 O ATOM 493 N LEU 85 −5.138 20.931 3.728 1.0028.47 N ATOM 494 CA LEU 85 −4.946 19.491 3.787 1.00 28.25 C ATOM 495 CBLEU 85 −3.572 19.113 3.231 1.00 29.46 C ATOM 496 CG LEU 85 −3.224 17.6283.151 1.00 31.51 C ATOM 497 CD1 LEU 85 −4.226 16.913 2.258 1.00 31.47 CATOM 498 CD2 LEU 85 −1.809 17.393 2.634 1.00 32.07 C ATOM 499 C LEU 85−5.125 19.006 5.229 1.00 27.80 C ATOM 500 O LEU 85 −5.923 18.105 5.5001.00 26.59 O ATOM 501 N PHE 86 −4.386 19.618 6.149 1.00 25.85 N ATOM 502CA PHE 86 −4.466 19.262 7.561 1.00 25.42 C ATOM 503 CB PHE 86 −3.15319.569 8.288 1.00 25.61 C ATOM 504 CG PHE 86 −1.984 18.745 7.833 1.0024.56 C ATOM 505 CD1 PHE 86 −0.723 18.972 8.362 1.00 25.69 C ATOM 506CD2 PHE 86 −2.158 17.687 6.955 1.00 25.22 C ATOM 507 CE1 PHE 86 0.34318.158 8.033 1.00 24.12 C ATOM 508 CE2 PHE 86 −1.096 16.866 6.620 1.0024.92 C ATOM 509 CZ PHE 86 0.156 17.100 7.161 1.00 25.70 C ATOM 510 CPHE 86 −5.626 19.871 8.342 1.00 25.22 C ATOM 511 O PHE 86 −5.984 19.3749.403 1.00 27.68 O ATOM 512 N GLY 87 −6.220 20.936 7.821 1.00 25.48 NATOM 513 CA GLY 87 −7.308 21.572 8.538 1.00 24.46 C ATOM 514 C GLY 87−6.846 22.301 9.793 1.00 24.27 C ATOM 515 O GLY 87 −7.577 22.374 10.7781.00 25.23 O ATOM 516 N VAL 88 −5.628 22.835 9.763 1.00 23.15 N ATOM 517CA VAL 88 −5.067 23.582 10.892 1.00 21.50 C ATOM 518 CB VAL 88 −4.18322.697 11.837 1.00 22.47 C ATOM 519 CG1 VAL 88 −5.014 21.573 12.444 1.0023.92 C ATOM 520 CG2 VAL 88 −2.985 22.136 11.082 1.00 21.60 C ATOM 521 CVAL 88 −4.200 24.704 10.341 1.00 19.98 C ATOM 522 O VAL 88 −3.544 24.5419.317 1.00 19.52 O ATOM 523 N PRO 89 −4.196 25.871 11.001 1.00 21.92 NATOM 524 CD PRO 89 −4.796 26.279 12.289 1.00 20.40 C ATOM 525 CA PRO 89−3.366 26.958 10.488 1.00 21.32 C ATOM 526 CB PRO 89 −4.009 28.16411.135 1.00 21.47 C ATOM 527 CG PRO 89 −4.109 27.664 12.548 1.00 21.05 CATOM 528 C PRO 89 −1.881 26.800 10.808 1.00 21.71 C ATOM 529 O PRO 89−1.029 27.350 10.114 1.00 20.80 O ATOM 530 N SER 90 −1.579 26.025 11.8451.00 22.01 N ATOM 531 CA SER 90 −0.197 25.803 12.257 1.00 24.39 C ATOM532 CB SER 90 0.475 27.107 12.704 1.00 25.95 C ATOM 533 OG SER 90 −0.19127.688 13.806 1.00 30.18 O ATOM 534 C SER 90 −0.076 24.728 13.329 1.0023.46 C ATOM 535 O SER 90 −1.048 24.407 14.010 1.00 25.46 O ATOM 536 NPHE 91 1.119 24.161 13.462 1.00 23.68 N ATOM 537 CA PHE 91 1.375 23.12614.463 1.00 22.17 C ATOM 538 CB PHE 91 0.869 21.755 14.002 1.00 22.00 CATOM 539 CG PHE 91 1.527 21.265 12.739 1.00 23.50 C ATOM 540 CD1 PHE 911.133 21.751 11.497 1.00 23.67 C ATOM 541 CD2 PHE 91 2.575 20.360 12.7981.00 21.96 C ATOM 542 CE1 PHE 91 1.775 21.342 10.339 1.00 22.42 C ATOM543 CE2 PHE 91 3.220 19.949 11.645 1.00 23.74 C ATOM 544 CZ PHE 91 2.81820.442 10.414 1.00 22.47 C ATOM 545 C PHE 91 2.862 23.032 14.812 1.0021.03 C ATOM 546 O PHE 91 3.700 23.714 14.225 1.00 22.64 O ATOM 547 NSER 92 3.162 22.177 15.781 1.00 18.50 N ATOM 548 CA SER 92 4.517 21.90916.234 1.00 17.97 C ATOM 549 CB SER 92 4.626 21.948 17.750 1.00 17.24 CATOM 550 OG SER 92 5.941 21.622 18.147 1.00 15.01 O ATOM 551 C SER 924.938 20.538 15.718 1.00 19.41 C ATOM 552 O SER 92 4.134 19.607 15.7111.00 16.66 O ATOM 553 N VAL 93 6.186 20.417 15.273 1.00 19.68 N ATOM 554CA VAL 93 6.676 19.142 14.766 1.00 20.81 C ATOM 555 CB VAL 93 8.05219.290 14.079 1.00 22.21 C ATOM 556 CG1 VAL 93 8.008 20.418 13.061 1.0021.22 C ATOM 557 CG2 VAL 93 9.134 19.522 15.119 1.00 20.18 C ATOM 558 CVAL 93 6.819 18.131 15.897 1.00 21.81 C ATOM 559 O VAL 93 7.136 16.96915.662 1.00 24.55 O ATOM 560 N LYS 94 6.579 18.568 17.127 1.00 22.80 NATOM 561 CA LYS 94 6.696 17.670 18.267 1.00 23.16 C ATOM 562 CB LYS 947.400 18.364 19.431 1.00 25.99 C ATOM 563 CG LYS 94 6.681 19.571 19.9661.00 29.97 C ATOM 564 CD LYS 94 7.486 20.206 21.088 1.00 34.85 C ATOM565 CE LYS 94 8.855 20.641 20.553 1.00 34.82 C ATOM 566 NZ LYS 94 9.71021.284 21.583 1.00 37.75 N ATOM 567 C LYS 94 5.343 17.088 18.669 1.0020.95 C ATOM 568 O LYS 94 5.253 16.252 19.570 1.00 19.08 O ATOM 569 NGLU 95 4.297 17.538 17.983 1.00 19.27 N ATOM 570 CA GLU 95 2.937 17.05518.212 1.00 18.96 C ATOM 571 CB GLU 95 1.919 18.145 17.873 1.00 18.81 CATOM 572 CG GLU 95 2.046 19.387 18.743 1.00 21.00 C ATOM 573 CD GLU 951.017 20.418 18.319 1.00 22.03 C ATOM 574 OE1 GLU 95 −0.095 20.44418.888 1.00 20.78 O ATOM 575 OE2 GLU 95 1.338 21.228 17.429 1.00 23.38 OATOM 576 C GLU 95 2.751 15.823 17.334 1.00 18.97 C ATOM 577 O GLU 952.008 15.853 16.359 1.00 17.30 O ATOM 578 N HIS 96 3.426 14.735 17.6981.00 20.49 N ATOM 579 CA HIS 96 3.374 13.503 16.919 1.00 22.53 C ATOM580 CB HIS 96 4.357 12.468 17.460 1.00 22.80 C ATOM 581 CG HIS 96 5.77812.924 17.429 1.00 22.99 C ATOM 582 CD2 HIS 96 6.331 14.078 16.987 1.0024.26 C ATOM 583 ND1 HIS 96 6.818 12.157 17.903 1.00 22.37 N ATOM 584CE1 HIS 96 7.952 12.818 17.754 1.00 25.34 C ATOM 585 NE2 HIS 96 7.68313.987 17.200 1.00 25.87 N ATOM 586 C HIS 96 2.016 12.865 16.703 1.0023.09 C ATOM 587 O HIS 96 1.741 12.375 15.611 1.00 25.80 O ATOM 588 NARG 97 1.153 12.871 17.712 1.00 23.57 N ATOM 589 CA ARG 97 −0.145 12.25317.509 1.00 22.11 C ATOM 590 CB ARG 97 −0.852 11.940 18.829 1.00 22.67 CATOM 591 CG ARG 97 −2.215 11.295 18.608 1.00 25.29 C ATOM 592 CD ARG 97−2.946 10.947 19.895 1.00 24.16 C ATOM 593 NE ARG 97 −4.238 10.34419.586 1.00 25.87 N ATOM 594 CZ ARG 97 −5.067 9.819 20.481 1.00 25.09 CATOM 595 NH1 ARG 97 −4.748 9.801 21.769 1.00 25.97 N ATOM 596 NH2 ARG 97−6.236 9.339 20.086 1.00 24.37 N ATOM 597 C ARG 97 −1.019 13.095 16.5961.00 20.82 C ATOM 598 O ARG 97 −1.837 12.563 15.849 1.00 22.99 O ATOM599 N LYS 98 −0.833 14.409 16.638 1.00 19.91 N ATOM 600 CA LYS 98 −1.61415.294 15.787 1.00 19.73 C ATOM 601 CB LYS 98 −1.487 16.749 16.228 1.0020.60 C ATOM 602 CG LYS 98 −2.252 17.723 15.331 1.00 23.23 C ATOM 603 CDLYS 98 −2.091 19.164 15.794 1.00 23.00 C ATOM 604 CE LYS 98 −2.61119.344 17.205 1.00 24.33 C ATOM 605 NZ LYS 98 −2.463 20.740 17.674 1.0025.97 N ATOM 606 C LYS 98 −1.181 15.160 14.331 1.00 18.95 C ATOM 607 OLYS 98 −2.013 15.142 13.423 1.00 18.06 O ATOM 608 N ILE 99 0.126 15.04714.120 1.00 16.71 N ATOM 609 CA ILE 99 0.664 14.935 12.777 1.00 18.14 CATOM 610 CB ILE 99 2.205 15.049 12.794 1.00 18.88 C ATOM 611 CG2 ILE 992.764 14.887 11.382 1.00 17.52 C ATOM 612 CG1 ILE 99 2.612 16.408 13.3721.00 16.52 C ATOM 613 CD1 ILE 99 4.102 16.610 13.445 1.00 18.05 C ATOM614 C ILE 99 0.252 13.632 12.105 1.00 19.52 C ATOM 615 O ILE 99 −0.09613.633 10.929 1.00 20.84 O ATOM 616 N TYR 100 0.278 12.520 12.836 1.0021.34 N ATOM 617 CA TYR 100 −0.140 11.258 12.234 1.00 21.76 C ATOM 618CB TYR 100 0.388 10.029 12.982 1.00 20.52 C ATOM 619 CG TYR 100 1.8809.828 12.897 1.00 21.94 C ATOM 620 CD1 TYR 100 2.456 9.359 11.724 1.0022.57 C ATOM 621 CE1 TYR 100 3.812 9.131 11.633 1.00 22.83 C ATOM 622CD2 TYR 100 2.708 10.070 13.982 1.00 22.53 C ATOM 623 CE2 TYR 100 4.0689.847 13.901 1.00 22.55 C ATOM 624 CZ TYR 100 4.613 9.374 12.724 1.0022.83 C ATOM 625 OH TYR 100 5.962 9.125 12.642 1.00 24.71 O ATOM 626 CTYR 100 −1.637 11.164 12.051 1.00 22.16 C ATOM 627 O TYR 100 −2.12010.375 11.240 1.00 20.79 O ATOM 628 N THR 101 −2.375 11.980 12.789 1.0022.43 N ATOM 629 CA THR 101 −3.818 11.949 12.659 1.00 20.93 C ATOM 630CB THR 101 −4.501 12.622 13.867 1.00 20.70 C ATOM 631 OG1 THR 101 −4.22011.862 15.049 1.00 21.16 O ATOM 632 CG2 THR 101 −6.013 12.703 13.6611.00 19.19 C ATOM 633 C THR 101 −4.176 12.684 11.382 1.00 20.33 C ATOM634 O THR 101 −4.980 12.199 10.583 1.00 19.32 O ATOM 635 N MET 102−3.551 13.843 11.186 1.00 20.81 N ATOM 636 CA MET 102 −3.803 14.66410.006 1.00 21.77 C ATOM 637 CB MET 102 −3.128 16.031 10.119 1.00 22.96C ATOM 638 CG MET 102 −3.563 16.875 11.305 1.00 25.26 C ATOM 639 SD MET102 −2.691 18.457 11.303 1.00 26.02 S ATOM 640 CE MET 102 −1.054 17.96111.843 1.00 25.10 C ATOM 641 C MET 102 −3.326 13.982 8.735 1.00 21.84 CATOM 642 O MET 102 −3.959 14.101 7.689 1.00 22.57 O ATOM 643 N ILE 103−2.215 13.259 8.828 1.00 20.60 N ATOM 644 CA ILE 103 −1.672 12.580 7.6641.00 20.83 C ATOM 645 CB ILE 103 −0.220 12.115 7.937 1.00 21.14 C ATOM646 CG2 ILE 103 0.262 11.190 6.832 1.00 20.31 C ATOM 647 CG1 ILE 1030.685 13.350 8.046 1.00 20.27 C ATOM 648 CD1 ILE 103 2.144 13.056 8.3231.00 20.70 C ATOM 649 C ILE 103 −2.568 11.414 7.248 1.00 20.54 C ATOM650 O ILE 103 −2.897 11.288 6.072 1.00 20.36 O ATOM 651 N TYR 104 −2.97410.569 8.194 1.00 20.06 N ATOM 652 CA TYR 104 −3.879 9.475 7.843 1.0018.57 C ATOM 653 CB TYR 104 −4.007 8.411 8.939 1.00 17.25 C ATOM 654 CGTYR 104 −2.810 7.512 9.145 1.00 18.10 C ATOM 655 CD1 TYR 104 −2.8366.214 8.649 1.00 19.08 C ATOM 656 CE1 TYR 104 −1.795 5.339 8.849 1.0018.97 C ATOM 657 CD2 TYR 104 −1.688 7.920 9.851 1.00 17.72 C ATOM 658CE2 TYR 104 −0.621 7.043 10.059 1.00 20.75 C ATOM 659 CZ TYR 104 −0.6915.746 9.551 1.00 20.88 C ATOM 660 OH TYR 104 0.323 4.835 9.749 1.0019.81 O ATOM 661 C TYR 104 −5.259 9.907 7.370 1.00 19.95 C ATOM 662 OTYR 104 −5.991 9.105 6.794 1.00 21.99 O ATOM 663 N ARG 105 −5.626 11.1627.602 1.00 17.59 N ATOM 664 CA ARG 105 −6.915 11.623 7.121 1.00 17.20 CATOM 665 CB ARG 105 −7.491 12.768 7.959 1.00 16.31 C ATOM 666 CG ARG 105−7.847 12.404 9.383 1.00 17.10 C ATOM 667 CD ARG 105 −8.413 13.60910.125 1.00 16.21 C ATOM 668 NE ARG 105 −8.799 13.284 11.495 1.00 16.48N ATOM 669 CZ ARG 105 −9.313 14.159 12.358 1.00 19.13 C ATOM 670 NH1 ARG105 −9.495 15.427 12.003 1.00 17.52 N ATOM 671 NH2 ARG 105 −9.676 13.76013.572 1.00 19.14 N ATOM 672 C ARG 105 −6.798 12.023 5.663 1.00 17.20 CATOM 673 O ARG 105 −7.789 12.336 5.021 1.00 16.63 O ATOM 674 N ASN 106−5.574 11.989 5.147 1.00 19.37 N ATOM 675 CA ASN 106 −5.302 12.366 3.7661.00 20.38 C ATOM 676 CB ASN 106 −4.507 13.663 3.704 1.00 20.43 C ATOM677 CG ASN 106 −5.251 14.812 4.339 1.00 20.58 C ATOM 678 OD1 ASN 106−6.143 15.390 3.723 1.00 23.31 O ATOM 679 ND2 ASN 106 −4.885 15.1605.562 1.00 17.80 N ATOM 680 C ASN 106 −4.659 11.281 2.919 1.00 21.95 CATOM 681 O ASN 106 −4.027 11.558 1.896 1.00 20.86 O ATOM 682 N LEU 107−4.823 10.042 3.361 1.00 21.77 N ATOM 683 CA LEU 107 −4.264 8.899 2.6661.00 21.73 C ATOM 684 CB LEU 107 −2.737 8.860 2.832 1.00 20.40 C ATOM685 CG LEU 107 −2.118 8.762 4.235 1.00 19.18 C ATOM 686 CD1 LEU 107−2.558 7.498 4.968 1.00 18.29 C ATOM 687 CD2 LEU 107 −0.602 8.811 4.1711.00 16.48 C ATOM 688 C LEU 107 −4.943 7.622 3.150 1.00 24.57 C ATOM 689O LEU 107 −5.476 7.574 4.261 1.00 26.59 O ATOM 690 N VAL 108 −4.9506.604 2.300 1.00 24.85 N ATOM 691 CA VAL 108 −5.535 5.314 2.637 1.0023.27 C ATOM 692 CB VAL 108 −6.585 4.884 1.594 1.00 25.05 C ATOM 693 CG1VAL 108 −6.727 3.374 1.592 1.00 26.97 C ATOM 694 CG2 VAL 108 −7.9235.523 1.917 1.00 23.65 C ATOM 695 C VAL 108 −4.435 4.259 2.704 1.0022.36 C ATOM 696 O VAL 108 −3.534 4.240 1.866 1.00 23.25 O ATOM 697 NVAL 109 −4.494 3.397 3.714 1.00 21.67 N ATOM 698 CA VAL 109 −3.504 2.3363.865 1.00 20.80 C ATOM 699 CB VAL 109 −3.455 1.801 5.316 1.00 19.45 CATOM 700 CG1 VAL 109 −2.544 0.583 5.389 1.00 17.84 C ATOM 701 CG2 VAL109 −2.941 2.892 6.256 1.00 18.14 C ATOM 702 C VAL 109 −3.787 1.1732.910 1.00 21.74 C ATOM 703 O VAL 109 −4.915 0.696 2.811 1.00 22.57 OATOM 704 N VAL 110 −2.754 0.739 2.195 1.00 22.61 N ATOM 705 CA VAL 110−2.871 −0.365 1.249 1.00 23.79 C ATOM 706 CB VAL 110 −1.791 −0.279 0.1581.00 22.99 C ATOM 707 CG1 VAL 110 −1.997 −1.377 −0.864 1.00 23.58 C ATOM708 CG2 VAL 110 −1.828 1.081 −0.495 1.00 24.01 C ATOM 709 C VAL 110−2.722 −1.703 1.961 1.00 24.53 C ATOM 710 O VAL 110 −3.639 −2.521 1.9441.00 28.90 O ATOM 711 OH2 WAT 901 6.275 31.112 15.565 1.00 15.47 O ATOM712 OH2 WAT 902 0.081 30.036 10.221 1.00 17.32 O ATOM 713 OH2 WAT 9030.127 15.519 19.408 1.00 7.93 O ATOM 714 OH2 WAT 904 7.030 2.992 5.9511.00 1.12 O ATOM 715 OH2 WAT 905 −8.260 9.003 4.793 1.00 16.56 O ATOM716 OH2 WAT 906 −0.287 23.410 17.398 1.00 20.45 O ATOM 717 OH2 WAT 90711.907 15.641 5.635 1.00 12.75 O ATOM 718 OH2 WAT 908 6.231 3.121 −2.8031.00 30.59 O ATOM 719 OH2 WAT 909 14.427 27.473 −1.777 1.00 32.06 O ATOM720 OH2 WAT 910 1.367 29.323 5.015 1.00 30.79 O ATOM 721 OH2 WAT 911−3.588 −4.814 0.678 1.00 24.03 O ATOM 722 OH2 WAT 912 3.828 6.836−11.268 1.00 35.90 O ATOM 723 OH2 WAT 913 8.152 2.609 8.906 1.00 34.73 OATOM 724 OH2 WAT 914 2.691 4.733 10.966 1.00 33.58 O ATOM 725 OH2 WAT916 −5.458 26.444 7.758 1.00 45.56 O ATOM 726 OH2 WAT 917 −8.076 13.27416.506 1.00 48.77 O ATOM 727 OH2 WAT 918 −7.491 17.267 10.217 1.00 40.88O ATOM 728 OH2 WAT 919 −3.101 7.362 −4.725 1.00 53.52 O ATOM 729 OH2 WAT920 1.887 19.751 −8.003 1.00 32.67 O ATOM 730 OH2 WAT 921 −8.379 15.505−1.840 1.00 19.94 O ATOM 731 OH2 WAT 922 12.181 19.059 −4.016 1.00 25.92O ATOM 732 OH2 WAT 923 −8.305 16.544 7.450 1.00 29.31 O ATOM 733 OH2 WAT924 −10.379 14.823 6.843 1.00 19.67 O ATOM 734 C1 SCH 999 14.935 15.34310.195 1.00 58.18 C ATOM 735 C2 SCH 999 13.897 15.023 11.252 1.00 58.15C ATOM 736 O1 SCH 999 14.212 14.616 12.373 1.00 58.19 O ATOM 737 N1 SCH999 12.629 15.230 10.832 1.00 57.26 N ATOM 738 C3 SCH 999 11.571 15.35311.839 1.00 54.45 C ATOM 739 C4 SCH 999 10.855 14.032 12.129 1.00 55.03C ATOM 740 O2 SCH 999 11.268 12.976 11.642 1.00 55.46 O ATOM 741 N2 SCH999 9.780 14.154 12.934 1.00 56.28 N ATOM 742 C5 SCH 999 9.102 12.95113.392 1.00 58.10 C ATOM 743 C6 SCH 999 10.025 12.199 14.360 1.00 59.43C ATOM 744 O3 SCH 999 10.530 11.115 14.068 1.00 59.32 O ATOM 745 N3 SCH999 10.203 12.847 15.527 1.00 61.53 N ATOM 746 C7 SCH 999 11.400 12.54216.310 1.00 62.93 C ATOM 747 C8 SCH 999 11.113 11.660 17.534 1.00 62.40C ATOM 748 O4 SCH 999 11.912 11.709 18.416 1.00 60.48 O ATOM 749 O5 SCH999 10.616 10.479 17.100 1.00 62.41 O ATOM 750 C9 SCH 999 10.586 16.47611.453 1.00 49.76 C ATOM 751 C10 SCH 999 12.105 13.839 16.718 1.00 64.21C ATOM 752 C11 SCH 999 13.629 13.756 16.707 1.00 65.67 C ATOM 753 C12SCH 999 14.196 14.646 17.803 1.00 66.24 C ATOM 754 O6 SCH 999 14.52615.775 17.594 1.00 66.36 O ATOM 755 O7 SCH 999 14.290 14.052 19.016 1.0065.17 O ATOM 756 C13 SCH 999 9.685 16.072 10.295 1.00 46.19 C ATOM 757C14 SCH 999 8.313 16.472 10.068 1.00 43.66 C ATOM 758 C15 SCH 999 7.87215.829 8.846 1.00 42.20 C ATOM 759 N4 SCH 999 8.951 15.079 8.368 1.0043.85 N ATOM 760 C16 SCH 999 10.006 15.227 9.219 1.00 44.78 C ATOM 761C17 SCH 999 7.411 17.318 10.780 1.00 43.03 C ATOM 762 C18 SCH 999 6.08517.525 10.294 1.00 39.51 C ATOM 763 C19 SCH 999 5.651 16.887 9.095 1.0039.12 C ATOM 764 C20 SCH 999 6.542 16.041 8.370 1.00 40.06 C ATOM 765CL1 SCH 999 4.034 17.133 8.522 1.00 34.69 CL ATOM 766 C21 SCH 999 8.77912.041 12.203 1.00 57.67 C ATOM 767 C22 SCH 999 7.820 13.335 14.135 1.0058.12 C ATOM 768 C23 SCH 999 6.793 13.968 13.201 1.00 56.83 C ATOM 769C24 SCH 999 7.749 12.676 11.271 1.00 57.14 C ATOM 770 C25 SCH 999 6.47713.064 12.015 1.00 56.55 C END

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A purified polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10and SEQ ID NO:
 12. 2. A compound represented by a structural formulawhich is selected from the group consisting of


3. A crystal comprising a complex between a polypeptide consisting ofthe amino acid sequence of SEQ ID NO: 10 and a compound represented bythe structural formula:

wherein said crystal is in space group P2₁2₁2₁ and comprises unit celldimensions a=37.999 Å, b=45.333 Å, c=63.999 Å, where α=β=Υ=90°.
 4. Acrystal comprising a complex between a polypeptide consisting of theamino acid sequence of SEQ ID NO: 6 and a compound represented by thestructural formula:

wherein said crystal is in space group P2₁2₁2₁ and comprises unit celldimensions a=41.1 Å, b=42.7 Å, c=53.777 Å, where α=β=Υ=90°.
 5. Thecrystal of claim 3 wherein the complex in said crystal is characterizedby structural coordinates set forth in Table
 3. 6. The crystal of claim4 wherein the complex in said crystal is characterized by structuralcoordinates set forth in Table
 4. 7. The polypeptide of claim 1comprising the amino acid sequence set forth in SEQ ID NO:
 6. 8. Thepolypeptide of claim 1 comprising the amino acid sequence set forth inSEQ ID NO:
 8. 9. The polypeptide of claim 1 comprising the amino acidsequence set forth in SEQ ID NO:
 10. 10. The polypeptide of claim 1comprising the amino acid sequence set forth in SEQ ID NO: 12.